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

²Department of Cytogenetics, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany

^aAuthor for correspondence

Amplification of the MYCN gene is found in a large proportion of neuroblastoma and considered as an adverse prognostic factor. To investigate the effect of ectopic MycN expression on the susceptibility of neuroblastoma cells to cytotoxic drugs we used a human neuroblastoma cell line harboring tetracycline-controlled expression of MycN. Neither conditional expression of MycN alone nor low drug concentrations triggered apoptosis. However, when acting in concert, MycN and cytotoxic drugs efficiently induced cell death. Apoptosis depended on mitochondrial permeability transition and activation of caspases, since the mitochondrion-specific inhibitor bongkrekic acid and the caspase inhibitor ZVAD-fmk almost completely abrogated apoptosis. Loss of mitochondrial transmembrane potential and release of cytochrome c from mitochondria preceded activation of caspase-8 and caspase-3 and cleavage of PARP. CD95 expression was upregulated by treatment with cytotoxic drugs, while MycN cooperated with cytotoxic drugs to increase sensitivity to CD95-induced apoptosis and enhancing CD95-L expression. MycN overexpression and cytotoxic drugs also synergized to induce p53 and Bax protein expression, while Bcl-2 and Bcl-X_L protein levels remained unchanged. Since amplification of MYCN is usually associated with a poor prognosis, these findings suggest that dysfunctions in apoptosis pathways may be a mechanism by which MycN-induced apoptosis of neuroblastoma cells is inhibited.

MycN; apoptosis; CD95; drugs; neuroblastoma

BA, bongkrekic acid; CD95-L, CD95 ligand; DiOC₆(3), 3,3'-dihexyloxacarbocyanide iodide; Doxo, Doxorubicin; ECL, enhanced chemiluminescence; FACS, fluorescence-activated cell-sorting; ICE, interleukin 1-converting enzyme; _m, mitochondrial transmembrane potential; PARP, poly(ADP-ribose) polymerase; Z-VAD.fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone

Introduction

Neuroblastoma is the second most common solid tumor of childhood arising from the peripheral sympathic nervous system (Berthold et al., 1990). Amplification of the MYCN gene is found in a large subset of neuroblastomas (Schwab et al., 1995) and is usually associated with resistance to therapy and a poor prognosis (Berthold et al., 1990). MYCN is a member of the family of MYC genes which encode transcription factors (Henriksson and Lüscher, 1996). A large body of experimental evidence suggests that proteins of the Myc family participate in the regulation of both cell growth and apoptosis (Henriksson et al., 1996; Packham et al., 1996). Enforced expression of MycN accelerates cell cycle progression (Lutz et al., 1996) and advances the malignant phenotype of neuroblastoma cells (Schweigerer et al., 1990). Targeted MycN expression has been described to cause neuroblastoma in transgenic mice (Weiss et al., 1997). In addition, MycN has been observed to induce apoptosis in B-lymphocytes when expressed from the immunoglobulin enhancer E in transgenic mice (Zörnig et al., 1995a).

Cytotoxic drugs irrespective of their intracellular target primarily act by inducing apoptosis in susceptible cells (Fisher et al., 1994). Activation of apoptosis pathways may involve triggering of CD95 ligand/receptor interaction that results in recruitment of the adaptor molecule FADD/MORT-1 and the receptor proximal caspase-8 (FLICE) to the activated CD95 receptor (Trauth et al., 1989; Oehm et al., 1992; Kischkel et al., 1995; Los et al., 1995; Peter et al., 1996; Muzio et al., 1996; Medema et al., 1997; Nagata, 1997). Activation of caspase-8 is followed by activation of downstream caspases (Nagata, 1997). Induction of CD95-L and upregulation of CD95 have been found in a variety of tumor cells including neuroblastoma after treatment with cytotoxic drugs (Friesen et al., 1996; Fulda et al., 1997a, 1998a,b; Müller et al., 1997). Blockade of CD95/CD95-L interaction by antagonistic antibodies to the receptor inhibited drug-induced apoptosis (Friesen et al., 1996; Fulda et al., 1997a, 1998a,b; Müller et al., 1997). Recent reports also suggest that the CD95 system may not be centrally involved in drug-induced apoptosis in some cell types (Eischen et al., 1997; Villunger et al., 1997).

Alterations of mitochondrial functions such as permeability transition have been found to play a major role in the apoptotic process (Kroemer et al., 1997a; Susin et al., 1997; Zamzami et al., 1996). Mitochondria undergoing permeability transition release apoptogenic proteins such as cytochrome c from the mitochondrial intermembrane space into the cytosol, where they can activate caspases and endonucleases (Kroemer et al., 1997a; Susin et al., 1997). Mitochondrial function during apoptosis seems to be controlled by the Bcl-2 family of proteins, which have been localized to intracellular membranes including the mitochondrial membrane (Kroemer et al., 1997b). Overexpression of the antiapoptotic molecules Bcl-2 and Bcl-X_L may inhibit apoptosis through their capacity to prevent mitochondrial permeability transition (Kroemer et al., 1997b), whereas overexpression of the pro-apoptotic protein Bax can induce mitochondrial permeability transition (Pastorino et al., 1998). In addition, Bcl-2 family members play key roles in the regulation of apoptosis by their ability to undergo both homo- and heterodimerization (Kroemer et al., 1997b).

Although Myc expression has been found to implement cells with programs for both proliferation and cell death (Packham et al., 1996), the role of Myc proteins in the cellular susceptibility to anticancer drugs is still obscure. Deregulated Myc expression has been reported to enhance tumor cell cytotoxicity in response to anticancer agents (Yu et al., 1997; Dong et al., 1997). On the other hand, overexpression of Myc proteins has been associated with resistance to cytotoxic drug treatment (Niimi et al., 1991). Therefore, to investigate the effect of ectopic MycN expression on drug-induced apoptosis in neuroblastoma, we used the human neuroblastoma cell line SHEP harboring tetracycline-controlled expression of MycN.

Results

MycN cooperates with cytotoxic drugs to induce apoptosis

To investigate the effect of MycN on the cellular susceptibility of neuroblastoma cells to cytotoxic drug treatment we used a synthetic inducible expression system on the basis of the tetracycline repressor of E. coli to reversibly express MycN in the neuroblastoma cell line SHEP in which expression of endogenous MycN is barely detectable (Lutz et al., 1996). Induced MycN alone or doxorubicin at low concentrations were unable to trigger apoptosis (Figure 1). However, induced MycN in concert with low concentrations of doxorubicin efficiently caused apoptosis (Figure 1). MycN also enhanced apoptosis found with higher concentrations of doxorubicin (Figure 1). To exclude that these findings were restricted to doxorubicin, experiments were also performed using cisplatinum. Again, induced MycN in combination with low concentrations of cisplatinum, which alone did not result in cell death, efficiently triggered apoptosis (data not shown). Induction of MycN expression in Tet-21/N cells was not influenced by treatment with cytotoxic drugs (data not shown). These findings indicate that enforced MycN expression cooperates with cytotoxic drugs to induce apoptosis in neuroblastoma cells.

MycN-induced apoptosis is mediated by activation of caspases

Proteins of the caspase family are involved in various forms of apoptosis (Nagata 1997). To investigate whether MycN-induced apoptosis was mediated by activation of the caspase cascade, we monitored the receptor proximal caspase FLICE (caspase-8), the downstream caspase CPP32 (caspase-3) and PARP, a prototype substrate of CPP32. Induced MycN in concert with doxorubicin resulted in cleavage of FLICE, CPP32 and PARP (Figure 2a). However, overexpression of MycN or low concentration of doxorubicin alone did not lead to activation of caspases (Figure 2a). Cisplatinum similarly cooperated with MycN in processing of caspases (data not shown). To further investigate whether apoptosis was the result of activation of caspases, we used the broad spectrum inhibitor of caspases ZVAD-fmk. Treatment with Z-fmk almost completely inhibited apoptosis induced by MycN in combination with doxorubicin demonstrating that apoptosis depends on activation of caspases (Figure 2b). These data suggest that the caspase cascade is differently regulated after drug treatment in MycN-overexpressing cells compared to parental cells and that selective activation of caspases may play an important role in the execution of an active cell death program.

To examine whether the difference in caspase activation was the result of altered expression levels of the adaptor protein FADD, which links proteases of the caspase cascade to cell surface receptors of the TNF/NGF family (Peter et al., 1996), Western blot analysis was performed. No difference in FADD protein expression was found upon MycN overexpression and/or doxorubicin treatment (data not shown), indicating that alterations in FADD protein levels do probably not account for the differences found in activation of caspases.

MycN and cytotoxic drugs synergize to activate the CD95 system

Since stimulation of death receptors of the TNF/NGF family such as CD95 leads to activation of the caspase cascade (Nagata, 1997) we next determined whether the CD95 system plays a role in apoptosis triggered by MycN and cytotoxic drugs. Incubation with doxorubicin resulted in upregulation of CD95 (Figure 3a). However, no increase in CD95 expression was detected after MycN activation (Figure 3a). Furthermore, both conditional expression of MycN alone and low drug doses alone increased the cellular susceptibility to apoptosis triggered by agonistic anti-APO-1 monoclonal antibodies (Figure 3b). Moreover, enforced expression of MycN in combination with drug treatment resulted in increased apoptosis compared to either treatment alone (Figure 3b). Enforced expression of MycN in concert with doxorubicin resulted in induction of CD95-L mRNA and protein (Figure 3c). However, when acting alone, conditional expression of MycN or doxorubicin at low concentrations had no detectable effect on CD95-L expression (Figure 3c). Taken together, this set of experiments suggests that MycN cooperates with cytotoxic drugs to activate the CD95 signaling pathway.

MycN and cytotoxic drugs cooperate to induce Bax and p53 expression

Proteins of the Bcl-2 family are known to positively and negatively regulate apoptosis (Kroemer et al., 1997a,b) and Bcl-2 has been reported to suppress Myc-induced apoptosis in fibroblasts (Bissonette et al., 1992). We therefore examined the role of Bcl-2 related proteins in the regulation of MycN-induced apoptosis. Increased Bax protein levels were found in cells overexpressing MycN after treatment with doxorubicin (Figure 4a). Overexpression of MycN or treatment with low concentrations of doxorubicin alone did not significantly alter the basal levels of Bax protein expression (Figure 4a). In addition, MycN overexpression and/or drug treatment had no detectable effect on Bcl-2 or Bcl-X_L protein levels (Figure 4a). Since p53 has been reported to mediate Myc-induced cell death (Hermeking and Eick, 1994) and to act as a transcriptional activator of the human BAX gene (Miyashita and Reed, 1995), we examined the potential role of p53 in modulating BAX expression and Myc-dependent cell death. After treatment with doxorubicin, p53 protein accumulated in cells overexpressing MycN (Figure 4a). However, no change in p53 protein expression was found after MycN activation or treatment with low drug concentrations alone (Figure 4a). Enhanced Bax protein levels were accompanied by increased BAX mRNA levels (Figure 4b) suggesting that Bax was regulated at the transcriptional level. These data suggest that MycN cooperates with doxorubicin in the accumulation of p53 protein and enhancement of Bax expression.

MycN cooperates with cytotoxic drugs to perturb mitochondrial functions

Mitochondria have been implicated in various systems of apoptosis (Kroemer et al., 1997a). Bax has been localized to the mitochondria membrane and is thought to promote apoptosis, at least in part, by interfering with mitochondrial functions (Pastorino et al., 1998; Wolter et al., 1997). Therefore, we next asked whether MycN-induced apoptosis following drug treatment was accompanied by alterations of mitochondrial functions. A drop in the mitochondrial transmembrane potential was noted in MycN-overexpressing cells following treatment with doxorubicin which was already detectable as early as 6 h after addition of the drug (Figure 5a). To see whether these mitochondrial alterations involve mitochondrial permeability transition, we tested the effect of bongkrekic acid (BA), a specific inhibitor of this pore (Susin et al., 1997; Zamzami et al., 1996). Addition of BA inhibited _m loss in response to MycN activation and drug treatment, indicating that mitochondrial alterations involved opening of PT pores (Figure 5b). Upon permeability transition, mitochondria have been reported to release apoptogenic proteins from the mitochondrial interspace into the cytosol that induce activation of caspases. Following doxorubicin treatment of MycN-overexpressing cells, there was a marked decline in mitochondrial cytochrome c levels, which was not found when MycN activation or low concentrations of doxorubicin were acting alone (Figure 5b). These data suggest that MycN and doxorubicin cooperate to trigger mitochondrial permeability transition and cytochrome c release.

Discussion

Amplification of MYCN is found in a large subset of patients with neuroblastoma and is considered to be one of the single most powerful prognostic factors indicating an adverse prognosis (Berthold, 1990). Studies on proteins of the Myc family revealed their roles in the regulation of both proliferation and cell death (Schwab et al., 1995) suggesting an integrated coregulation of proliferative and apoptotic signal transduction pathways (Henriksson and Lüscher, 1996). In the present study, we therefore analysed the effect of deregulated MycN expression on the susceptibility of neuroblastoma cells to anticancer drugs using a human neuroblastoma cell line with tetracyline-controlled expression of MycN. Enforced expression of MycN or low concentrations of cytotoxic drugs barely triggered apoptosis when acting alone. However, conditional expression of MycN in concert with cytotoxic drugs efficiently induced apoptosis indicating a strong cooperation.

MYCN synergized with anticancer drugs to induce apoptosis along several different pathways, e.g. by increasing expression levels of Bax mRNA and protein. Induction of Bax may be mediated by p53 protein, which accumulated in response to enforced MycN expression in combination with drug treatment (Figure 4a). p53 has been described to act as a transcriptional activator of the human BAX gene (Miyashita and Reed, 1995). Alternatively, MycN may directly lead to transcriptional activation of the BAX gene as there are several potential binding sites for Myc proteins in the BAX promotor (Miyashita and Reed, 1995). Bax can promote apoptosis through heterodimerization with anti-apoptotic Bcl-2 family members (Kroemer et al., 1997). Upon drug treatment of MycN-overexpressing cells Bax levels increased without any significant change in Bcl-2 or Bcl-X_L expression, thereby changing the ratio of pro-apoptotic to anti-apoptotic Bcl-2 family members in favor of apoptosis. In addition, Bax has recently been reported to exert its pro-apoptotic effect by interfering with mitochondrial functions (Pastorino et al., 1998). Bax has been localized to the mitochondrial membrane or can redistribute from the cytosol to mitochondria upon induction of apoptosis (Wolter et al., 1997). Mitochondrial perturbations contributed to the cooperative effect of MycN and cytotoxic drugs, since inhibition of mitochondrial permeability transition by the mitochondrion-specific inhibitor bongkrekic acid blocked doxorubicin-induced loss of the mitochondrial membrane potential and apoptosis. In addition, release of cytochrome c from the mitochondria to the cytosol, which can induce cleavage of caspases, was only found when MycN was acting together with cytotoxic drugs.

Moreover, MycN and cytotoxic drugs cooperated to activate the CD95 system. CD95 was upregulated in response to cytotoxic drug treatment in line with previous reports (Fulda et al., 1997a, 1998a,b; Müller et al., 1997). In addition, MycN and doxorubicin synergized to increase the cellular susceptibility to CD95-induced apoptosis indicating that MycN acted downstream of the CD95 receptor. The increase in Bax protein expression, which we found in MycN-overexpressing cells upon drug treatment, might contribute to the enhanced sensitivity to the CD95 death signal, since overexpression of Bax has been shown to increase sensitivity to CD95-induced apoptosis (Bargou et al., 1996). MycN and cytotoxic drugs also synergized to induce CD95-L expression. At present, the molecular events regulating CD95-L expression in response to cytotoxic drug treatment are only partially understood and may be mediated by activation of the JNK/SAPK-dependent stress pathway (Herr et al., 1997; Faris et al., 1998). Taken together, our findings demonstrate that cooperation between MycN overexpression and drug treatment may, at least in part, be attributed to activation of the CD95 signaling pathway. Apoptosis induced by Myc has recently been linked to the CD95 system by demonstrating that c-Myc sensitizes rat fibroblasts for CD95-mediated apoptosis and that the adaptor molecule FADD is involved in c-Myc-induced apoptosis (Hueber et al., 1997). However, there is also a recent report in FADD knock-out mice showing that FADD is not required for c-Myc-induced apoptosis (Yeh et al., 1998). Previous work in our laboratory and data from the literature demonstrated that enforced expression of MycN not only enhanced the susceptibility to CD95-mediated apoptosis, but also increased the cellular response towards other death-inducing ligands of the TNF/NGF family such as TRAIL and TNF (Lutz et al., 1998; Klefstrom et al., 1994, 1997; Janicke et al., 1994). In addition, MycN activation increased the sensitivity to cytokines such as IFN (Lutz et al., 1998). Thus, Myc proteins may enhance the susceptibility towards various death signals, much in the same way as they increase the effect of various growth factors.

Furthermore, MycN-dependent apoptosis in response to cytotoxic drugs correlated with a selective activation of the caspase cascade. The receptor proximal caspase-8 might be processed at the receptor level upon activation of the CD95 system during MycN-dependent apoptosis. Caspase-8 has previously been reported to be cleaved at the activated CD95 death-inducing signaling complex (Medema et al., 1997). In addition, we recently reported that both the receptor proximal caspase-8 and the downstream caspase-3 can be processed by mitochondria undergoing apoptosis (Fulda et al., 1997b; Scaffidi et al., 1998). Since we found a decrease in mitochondrial cytochrome c levels upon drug treatment in MycN-overexpressing cells, but not in cells without MycN activation, the release of apoptogenic proteins from mitochondria such as cytochrome c are probably also involved in activation of caspases and cell death.

Taken together, MycN and cytotoxic drugs seem to cooperate to trigger apoptosis at several levels involving alterations in mitochondrial functions, induction of Bax expression and activation of the CD95 system resulting in cleavage of caspases and cell death. Our findings may have profound implications for tumorigenesis and treatment response of neuroblastoma given the frequent overexpression of MYCN in patients with neuroblastoma and its adverse prognostic impact. The potent apoptotic activity of deregulated MycN in response to cytotoxic drug treatment may indicate that neuroblastoma cells with MYCN amplification can resist treatment only when additional dysfunction in apoptosis signaling pathways are acquired. Oncogenic synergy between the absence of CD95 and overexpression of Myc has previously been reported in vivo (Zörnig et al., 1995b). In addition, for c-Myc and Bcl-2, oncogenic cooperation has been shown to result from acquired anti-apoptotic mechanisms, since Bcl-2 blocks c-Myc-induced apoptosis while leaving the proliferative potential of c-Myc unaffected (Bissonette et al., 1992). Thus, dysfunctions in apoptosis pathways may be a mechanism by which MycN-induced apoptosis of neuroblastoma cells is suppressed.

Materials and methods

Drugs

Doxorubicin (Farmitalia, Milano, Italy) and cisplatinum (Sigma, Deisenhofen, Germany) were provided as pure substances and dissolved in sterile water prior to each experiment (1 mg/ml).

Cell culture and treatment with antibodies, ZVADfmk or bongkretic acid

The generation, characterization and culture of the MycN-inducible cell line SHEP Tet-21/N has previously been described (Lutz et al., 1996). Induction of MycN expression by removal of tetracycline from the growth medium was carried out shortly before culture reached confluence as apoptosis was most pronounced in confluent cultures. Anti-APO-1 IgG₃ (anti-CD95) antibody was added at a concentration of 1 g/ml. The broad spectrum tripeptide inhibitor of caspases ZVAD-fmk (Enzyme Systems Products, Dublin, USA) was used at a concentration of 60 M and BA at a concentration of 50 M (kindly provided by Dr Duine, University of Delf, The Netherlands).

Determination of apoptosis

Cells were plated at a density of 5´10⁴ cells/cm² and incubated for indicated times with doxorubicin, cisplatinum, VP-16 or Anti-APO-1 IgG₃ antibody. Cells were harvested by trypsinization using 0.05% trypsin and 0.02% EDTA without Ca²⁺ and Mg²⁺ (Life Technologies, Inc.). Quantification of DNA fragmentation was performed by FACS analysis of propidium iodide stained nuclei as previously described (Nicoletti et al., 1991) using CELLQuest software (Becton Dickinson, Heidelberg, Germany).

Detection of APO-1 (CD95) expression

Cells were stained with anti-APO-1 (anti-CD95) IgG₁ monoclonal antibody (1 g/ml) for 45 min at 4°C followed by goat anti-mouse IgG-phycoerythrin (Immunotech, Hamburg, Germany) for 30 min at 4°C. FII23 IgG3 antibody was used as isotype matched non-binding antibody to control unspecific bindings. Cells were analysed by flow cytometer using CELLQuest software.

Determination of mitochondrial membrane potential

For determination of mitochondrial membrane potential, cells (5´10⁵/ml) were incubated with 3,3'-dihexyloxacarbocyanide iodide (DiOC₆(3), 40 nM, Molecular Probes, Inc., Eugene, OR, USA) for 15 min at 37°C and analysed on a flow cytometer (FACScan).

RT - PCR for CD95-L mRNA

Total RNA was prepared using the Qiagen total RNA kit (Qiagen, Hilden, Germany). RNA was converted to cDNA by reverse transcription and amplified for 38 cycles by PCR in a thermocycler (Stratagene, Heidelberg, Germany) using the Gene Amplification RNA - PCR kit (Perkin Elmer, Branchburg, NJ, USA) following the manufacturer's instructions. A 500-base pair fragment of CD95-L was amplified using primer 5'-ATGTTTCAGCTCTTCCACCTACAGA-3' and 5'-CCAGAGAGAGCTCAGATACGTTGAC-3' according to the sequence of human CD95-L. Expression of -actin (MWG-Biotech, Ebersberg, Germany) was used as a standard for RNA integrity and equal gel loading. PCR-reaction products were run at 60 V for 2 h on a 1.5% agarose gel stained with ethidium bromide and visualized by UV illumination.

Preparation of mitochondria

For isolation of mitochondria, cells (3´10⁸ per sample) were washed twice with ice-cold PBS and resuspended with five volumes of buffer A (50 mM Tris buffer, 1 mM EGTA, 5 mM 2-mercaptoethanol, 0.2% BSA, 10 mM KH₂PO₄, pH 7.6, 0.4 M sucrose) and allowed to swell on ice for 20 min. Cells were homogenized with 30 strokes of a teflon homogenizer and centrifuged at 4000 g for 1 min at 4°C. The supernatants were further centrifuged at 10 000 g for 10 min at 4°C and the resulting pellets were resuspended in buffer B (10 mM KH₂PO₄. pH 7.2, 0.3 mM mannitol, 0.1% BSA). Mitochondria were separated by sucrose gradient (lower layer: 1.6 M sucrose, 10 mM KH₂PO₄, pH 7.5, 0.1% BSA; upper layer: 1.2 M sucrose, 10 mM KH₂PO₄, pH 7.5, 0.1% BSA). Interphases containing mitochondria were washed with buffer B at 18 000 g for 10 min at 4°C and the resulting mitochondrial pellets were resuspended in buffer B. The protein concentration of mitochondria was determined by the Bradford method.

Western blot analysis

Cells were lysed for 30 min at 4°C in PBS with 0.5% Triton X (Serva, Heidelberg, Germany) and 1 mM PMSF (Sigma, Deisenhofen, Germany) followed by high-speed centrifugation. Membrane proteins were eluted in buffer containing 0.1 M glycine, pH 3.0 and 1.5 M Tris, pH 8.8. Protein concentration was assayed using bicinchoninic acid (Pierce, Rockford, IL, USA). Forty g protein per lane was separated by 12% SDS - PAGE and electroblotted onto nitrocellulose (Amersham, Braunschweig, Germany). Equal protein loading was controlled by Ponceau red staining of membranes. After blocking for 1 h in PBS supplemented with 2% BSA (Sigma) and 0.1% Tween 20 (Sigma), immunodetection of FLICE, CPP32, PARP, Bax, Bcl-x, Bcl-2 and p53 protein was done using mouse anti-FLICE monoclonal antibody C15 (Scaffidi et al., 1997; 1 : 5 dilution of hybridoma supernatant), mouse anti-CPP32 monoclonal antibody (1 : 1000, Transduction Laboratories, Lexington, KY, USA), rabbit anti-PARP polyclonal antibody (1 : 10000, Enzyme Systems Products), rabbit anti-Bax polyclonal antibody (1 : 500, Calbiochem, Bad Soden, Germany), rabbit anti-Bcl-x polyclonal antibody (1 : 1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Bcl-2 monoclonal antibody (1 : 1000, Santa Cruz Biotechnology), mouse anti-p53 monoclonal antibody (1 : 1000, Transduction Laboratories) and goat anti-mouse IgG or goat anti-rabbit IgG (1 : 5000, Santa Cruz Biotechnology). ECL (Amersham) was used for detection.

Acknowledgements

This work has been partially supported by grants from the Deutsche Forschungsgemeinschaft, the Bundesministerium für Forschung and Technologie, Bonn, the Tumor Center Heidelberg/Mannheim, The Deutsche Leukämieforschungshilfe (to K-MD), the Dr Mildred Scheel Stiftung and the Fördergemeinschaft Kinderkrebs-Neuroblastom-Forschung e.V. (to MS).

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Figure 1 MycN overexpression increases sensitivity to doxorubicin-induced apoptosis. Cells were treated with 0.1 g/ml doxorubicin for 48 h in the presence (black bars) or absence (white bars) of MycN expression. Apoptosis was determined by FACS analysis of propidium iodide stained nuclei. Data are the mean of triplicates with standard deviations of less than 10%. Similar results were obtained in three separate experiments

Figure 2 Activation of caspases. (a) MycN and doxorubicin cooperate to activate caspases. Cells were treated with 0.1 g/ml doxorubicin for indicated times in the presence (+MYCN) or absence (-MYCN) of MYCN expression. Forty g protein per lane isolated from cell lysates were separated by 15% SDS - PAGE. Immunodetection of FLICE, CPP32 and PARP protein was performed by mouse anti-FLICE monoclonal antibody, mouse anti-CPP32 monoclonal antibody or rabbit anti-PARP polyclonal antibody and ECL. Processing of FLICE which was detected as a double band corresponding to two FLICE isoforms (caspase-8/a and 8/b) resulted in the p43 and p41 cleavage intermediates derived from caspase-8/a and 8/b, respectively, and the p18 active subunit. (b) Inhibition of apoptosis by the caspase inhibitor ZVAD-fmk. MycN overexpressing cells were treated with 0.1 g/ml doxorubicin for 48 h in the presence (white bars) or absence (black bars) or 50 M ZVAD.fmk. Apoptosis was determined by FACS analysis of propidium iodide stained nuclei. Percentage of specific apoptosis was calculated as described in Figure 1a. Similar results were obtained in three separate experiments

Figure 3 MycN and doxorubicin cooperate in activating the CD95 system. (a) Induction of CD95 expression. Cells were treated with 0.1 g/ml doxorubicin (Doxo) for 24 h in the presence or absence of MYCN expression, stained with mouse anti-APO-1 monoclonal antibody followed by phycoerythrin-conjugated anti-mouse IgG antibody and analysed by flow cytometry using CELLQuest software. Thick line: untreated or treated cells stained with anti-APO-1 antibody, dotted line: control cells stained with isotype-matched antibody, thin line: unstained cells. Fluorescence intensity (abscissa) is plotted against cell number (ordinate). Similar results were obtained in three separate experiments. (b) Induction of CD95-L mRNA. Cells were treated with 0.1 g/ml doxorubicin for indicated times in the presence (+MYCN) or absence (-MYCN) of MYCN expression. CD95-L mRNA expression was determined by RT - PCR. Expression of -actin was used to control RNA integrity and equal gel loading. Forty g protein of cell lysates per lane were separated by 12% SDS - PAGE CD95-L protein was detected as a 37 kDa band by mouse anti-CD95-L monoclonal antibody and ECL. (c) Increased sensitivity to APO-1-induced apoptosis. Cells were treated for 24 h with 1 g/ml APO-1 monoclonal antibody with or without doxorubicin pretreatment (0.1 g/ml) for 24 h and in the presence (+MYCN) or absence (-MYCN) of MYCN expression. Apoptosis was determined by FACS analysis of propidium iodide stained nuclei. Percentage of specific apoptosis was calculated as described in Figure 1. Similar results were obtained in three separate experiments

Figure 4 Modulation of Bcl-2 related proteins and p53. (a) Induction of Bax and p53 protein. Cells were treated with 0.1 g/ml doxorubicin for indicated times in the presence (+MYCN) or absence (-MYCN) of MYCN expression. Forty g protein per lane isolated from cell lysates were separated by 12% SDS - PAGE. Immunodetection of Bax, Bcl-2, Bcl-X_L, and p53 protein was performed by rabbit anti-Bax polyclonal antibody, mouse anti-Bcl-2 monoclonal antibody, rabbit anti-Bcl-X_L polyclonal antibody or mouse anti-p53 monoclonal antibody and ECL. (b) Induction of BAX mRNA. Cells were treated with 0.1 g/ml doxorubicin for 12 h in the presence (+MYCN) or absence (-MYCN) of MYCN expression. BAX mRNA expression was determined by RT - PCR. Expression of -actin was used to control RNA integrity and equal gel loading

Figure 5 Perturbations of mitochondrial functions. (a) Loss of _m. Cells were treated with 0.1 g/ml doxorubicin for indicated times in the presence (+MYCN) or absence (-MYCN) of MYCN expression. _m was determined by staining cells with the potential-sensitive fluorochrome DiOC₆(3). (b) Inhibition of _m loss by bongkrekic acid (BA). MYCN-overexpressing cells were treated with 0.1 g/ml doxorubicin for 24 h in the presence (+BA) or absence (-BA) of 50 M BA. _m was determined by staining cells with the potential-sensitive fluorochrome DioOC₆(3). (c) Doxorubicin-induced release of cytochrome c from mitochondria. Cells were treated with 0.1 g/ml doxorubicin for 24 h in the presence (+MYCN) or absence (-MYCN) of MYCN expression. Mitochondria were prepared as described in Materials and methods. Five g protein per lane were separated by 15% SDS - PAGE. Immunodetection of cytochrome c was performed by mouse monoclonal anti-cytochrome c antibody and ECL