Receptor Activator of Nuclear Factor B Ligand Plays a Nonredundant Role

[CANCER RESEARCH 63, 1772–1775, April 15, 2003] Advances in Brief

Receptor Activator of Nuclear Factor ␬B Ligand Plays a Nonredundant Role in Doxorubicin-induced Apoptosis1

Ingo Mu¨ller,2 Stefan M. Pfister, Ulrike Grohs, Janine Zweigner, Rupert Handgretinger, Dietrich Niethammer, and Gernot Bruchelt University Children’s Hospital Tu¨bingen, Department of General Pediatrics, Hematology and Oncology, 72076 Tuebingen, Germany [I. M., S. M. P., U. G., D. N., G. B.], and St. Jude Children’s Research Hospital, Memphis, Tennessee 38105 [J. Z., R. H.]

Abstract modulation of the incoming death signal. During doxorubicin-induced apoptosis, not only membrane receptors and their ligands are differen- Doxorubicin induces apoptosis in a variety of cells. We investigated the tially expressed but also downstream signaling molecules or transcription expression and function of various tumor necrosis factor (TNF)␣-homologues factors (8). One of the most controversial key players is NF-␬B. On one and their receptors. CEM cells did not differentially express any one of the TNF␣-homologous receptors investigated nor TNF-related apoptosis-induc- hand, it is most often considered an antiapoptotic transcription factor (9), ing ligand or TNF-related weakly apoptosis-inducing ligand (TWEAK) in the but on the other hand, a cleavage product generated from NF-␬Bby presence of doxorubicin. In addition to CD95 ligand, however, receptor activated caspase 8 can facilitate the completion of the apoptotic program activator of nuclear factor ␬B ligand (RANKL) was strongly up-regulated. (10). Moreover, in the promotor region of CD95L, several putative- Doxorubicin-induced apoptosis was greatly suppressed in the presence of binding sites for NF-␬B have been identified that are necessary to either neutralizing antibody or RANK-Fc fusion protein. Moreover, neutral- transactivate CD95L expression in drug-induced apoptosis (11). NF-␬B izing RANKL also prevented cytochrome c release from mitochondria. is involved in the regulation of well over 100 genes, and there are many RANKL alone was unable to induce significant levels of apoptosis in CEM ways documented to activate this transcription factor, one of which is cells. However, doxorubicin-induced apoptosis was increased 2-fold when > ␣ exogenous RANKL was added. Therefore, RANKL is necessary but not engagement of the TNF -homologue RANK by its cognate ligand sufficient to account for early doxorubicin-induced apoptosis in CEM cells. RANKL (12). RANKL is mainly expressed in osteoclasts and dendritic This finding suggests improved chemotherapeutic efficiency of the anthracy- cells (13). RANKL-deficient mice display a most striking phenotype, in clin against susceptible malignant cells in the presence with RANKL. that they are lacking all lymph nodes and have severe osteopetrosis (14). In the immune system, RANKL participates in T-cell activation (15) and Introduction maturation of dendritic cells (12). The structural similarity of RANKL ␣ Cytostatic drugs exert their toxic effects on malignant cells by a variety with other members of the TNF -family prompted us to investigate its of mechanisms, many of which lead to apoptotic cell death. One of the role in chemotherapy-induced cell death. We found that its expression is proposed mechanisms is the up-regulation of CD95L and subsequent induced by incubation of the T-lymphoblastic cell line CEM with doxo- autocrine suicide by its binding to CD95 expressed by the same cell (1). rubicin at pharmacologically relevant concentrations. Neutralizing In a growing body of literature investigating the role of CD95 in chemo- ␣RANKL antibody, as well as RANK-Fc fusion protein, inhibited doxo- therapy-induced apoptosis, several experiments were carried out in which rubicin-induced apoptosis in this system. Exogenous RANKL increased the interaction of CD95 with CD95L or either molecule was eliminated the rate of apoptosis induced by doxorubicin Ͼ2-fold. This shows that the and apoptosis could still be induced (2). We have reported earlier that toxicity of doxorubicin in this system is limited by the availability of unlike in other settings, de novo protein synthesis is necessary for doxo- RANKL, and administration of exogenous RANKL could increase the rubicin-induced apoptosis to occur (3). Obviously, molecules other than efficiency of anthracyclin-based chemotherapy. CD95/CD95L are contributing to the apoptotic effect of cytostatic drugs, such as doxorubicin, one of the most frequently used anthracycline Materials and Methods derivatives in this experimental setting (4). In recent years, several new TNF␣3 homologues and their corresponding receptors have been cloned Cell Culture, Antibodies, and Induction of Apoptosis. CCRF-CEM cells (reviewed in Ref. 5). This raised the possibility that there may be cell were obtained from the American Type Tissue Culture Collection (Manassas, VA). Cells were cultured at 37°C under 5% CO in RPMI 1640 supplemented with type-dependent differences, and numerous TNF␣-homologues, which 2 10% (volume for volume) FCS, 0.5 mML-glutamine, 100 I.E./ml penicillin, and bind to receptors containing death domains, have been investigated in this 100 ␮g/ml streptomycin. Experiments were carried out with cells from exponen- context (6). One of these turned out to be especially interesting, because tially growing cultures never exceeding a density of 5 ϫ 106 cells/ml. doxorubicin was found to sensitize breast cancer cells to TRAIL-induced Doxorubicin was added to a final concentration of 1 ␮M where indicated. apoptosis (7). Another layer of complexity is added by intracellular ␣ hRANKL M365 and M366 antibodies, as well as RANK-Fc, were kindly provided by Dr. D. Anderson of Immunex Corp. (Seattle, WA). Soluble Received 4/22/02; accepted 2/21/03. recombinant human RANKL, neutralizing ␣CD95L clone H11 and agonistic The costs of publication of this article were defrayed in part by the payment of page ␣CD95 clone DX2 were obtained from Alexis (Laeufelingen, Switzerland). charges. This article must therefore be hereby marked advertisement in accordance with All antibodies were used in a final concentration of 3 ␮g/ml. 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by the fortu¨ne program of the University Hospital Tu¨bingen, RT-PCR. Total RNA was isolated using the High Pure RNA Isolation Kit Germany. (Boehringer Mannheim, Mannheim, Germany) according to the manufactur- 2 To whom requests for reprints should be addressed, at University Children’s Hos- er’s instructions. RNA content was assessed by photometry at 260 nm, and 1 pital, Hoppe-Seyler-Str. 1, 72076 Tuebingen, Germany. Phone: 49-7071-2981323; Fax: ␮ 49-7071-295482; E-mail: [email protected]. g of RNA was subjected to reverse transcription. Specific primers were 4 3 The abbreviations used are: TNF, tumor necrosis factor; TRAIL, tumor necrosis designed using Primer 3, and GAPDH amplification served as internal stand- factor-related apoptosis-inducing ligand; NF-␬B, nuclear factor ␬B; RANK, receptor ard. PCR conditions for internal standard and samples: (a) 30 cycles with 30 s activator of nuclear factor ␬B; RT-PCR, reverse transcription-PCR; RANKL, receptor activator of nuclear factor ␬B ligand; GAPDH, glyceraldehyde-3-phosphate dehydrogen- ase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. 4 Internet address: http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi. 1772

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. TNF〈-HOMOLOGUE RANKL PLAYS A ROLE IN APOPTOSIS at 95°C; (b)60sat60°C; and (c)90sat72°C. PCR products were analyzed by PAGE and densitometry. Apoptosis. Apoptosis was detected by the Annexin-V Kit (Boehringer Mannheim) according to the manufacturer’s protocol. Cells were analyzed by flow cytometry on a FACScan using the CellQuest software (Becton Dickin- son, San Jose, CA). In addition to fluorescence-activated cell sorter analysis, samples were subjected to the Cell Death Detection ELISA (Boehringer Mannheim) according to the standard protocol provided by the manufacturer. Cell Viability. Cell survival after treatment with doxorubicin and/or neutralizing agents was measured by MTT as described earlier (16). All samples were assayed in triplicate. Cytochrome c Release. Cytochrome c was detected in the cytosolic fraction of CEM cells by Western blot according to a protocol published earlier (17). Briefly, 3 ϫ 106 CEM cells were pelleted and resuspended in 500 ␮l of digitonin lysis buffer consisting of 210 mMD-mannitol, 70 mM sucrose, 10 mM HEPES, 200 ␮M EGTA, 5 mM succinate, 0.15% (volume for volume) BSA, and 20 ␮g/ml digitonin. After incubation for 5 min at 4°C, cells were pelleted again, and the supernatant was transferred into a fresh microcentrifuge tube. After centrifugation at 13,000 ϫ g for 10 min at 4°C, the supernatant was combined with 600 ␮lof 10% (w/v) trichloric acid. The precipitated proteins after incubation at Ϫ20°C for 30 min were collected by centrifugation at 13,000 ϫ g for 10 min at 4°C. The pellet was resuspended in 50 ␮l of Laemmli loading buffer, boiled for 5 min, and separated on a 12% SDS-PAGE gel. Chemicals were obtained from Sigma- Aldrich (Taufkirchen, Germany). Proteins were blotted overnight at 4°C onto a Hybond polyvinylidene difluoride membrane (Amersham-Pharmacia, Piscataway, NJ) and detected using ␣-hCyt c-mAb 7H8 2C12 (Alexis, Gru¨nberg, Germany) and enhanced chemiluminescence reagents (Amersham-Pharmacia, Freiburg, Ger- many) according to the manufacturer’s protocol. NF-␬B Phosphorylation. Cells were cultured at a density of 5 ϫ 105/ml in 10% FCS supplement RPMI 1640 in the presence of 3 ␮g/ml RANKL and 1 Fig. 1. Semiquantitative RT-PCR of RNA extracted from CEM cells after culture in the ␮M doxorubicin, where appropriate. Cells were harvested after 3 or6hof presence of 1 ␮M doxorubicin for the indicated time periods. CEM cells not only incubation, washed in PBS (pH 7.4), and lysed in Laemmli buffer. Lysates up-regulate CD95L (a) but also RANKL (b) relative to GAPDH. This is the representative result of three experiments. were separated on a 12% (w/v) SDS gel by electrophoresis and transferred to polyvinylidene difluoride membrane overnight at 20 V using a MiniProtean3 cell (Bio-Rad). The phosphorylation-specific antibody Phospho-NF-␬B p65 (Ser536; Cell Signaling Technology, Beverly, MA) was used as primary pensated for by CD95L. Consequently, RANKL acts independent or antibody at a 1:2000 dilution in Tris(hydroxymethyl)aminomethane-buffered upstream of CD95L. saline with 0.1% (v/v) Tween-20 (TBST) and polyclonal ␣-rabbit Ig-horse- RANKL Amplifies Doxorubicin-induced Apoptosis. As RANKL radish peroxidase conjugate (Santa Cruz Biotechnology, Santa Cruz, CA) as plays a pharmacologically relevant role in doxorubicin-induced apoptosis detection reagent at a 1:5000 dilution in TBST. Visualization was done using in CEM cells, we asked if the recombinant protein itself is able to induce enhanced chemiluminescence reagents (Amersham-Pharmacia) according to apoptosis in these cells. Therefore, we incubated CEM cells with recom- the manufacturer’s protocol by light sensitive film, as well as by the Storm Յ ␮ device (Amersham-Pharmacia), to facilitate densitometric assessment. binant human RANKL at concentrations of 10 g/ml. Cells were then analyzed for characteristic morphological apoptotic changes, as well as Results for exposure of phosphatidylserine on the outer leaflet of the cellular membrane. Surprisingly, CEM cells did not become apoptotic in the Expression of TNF␣-Homologues. To assess the influence of presence of rhRANKL (Fig. 3). This experiment rules out nonspecific doxorubicin on expression of TNF␣-homologues and their receptors, we binding of this TNF␣-homologue to receptors like CD95 or the TRAIL used RT-PCR analysis of RNA extracted from CEM cells after incuba- receptor DR5 in the concentrations under investigation. There was no tion for 12 h in the presence of 1 ␮M doxorubicin. Specific primers were change in the cell cycle analysis of CEM in the presence of 1 ␮g/ml designed, and GAPDH served as an internal standard. All investigated rhRANKL or 3 ␮g/ml rhRANKL either (data not shown). The incubation mRNAs (TRAIL, TRAIL-R1, the splice forms TRICK-A and TRICK-B of CEM cells with both RANKL and doxorubicin, however, resulted in of TRAIL-R2, and TRAMP) were detected in CEM cells. Neither TRAIL a 2.2-fold increase of apoptosis. Obviously, RANKL plays a nonredun- nor one of its receptors showed significant variation in expression levels dant role in this system but is not sufficient to account for the proapo- (data not shown). RANK was not significantly modulated in expression ptotic effect of doxorubicin. either; however, its cognate ligand RANKL was increased 5-fold (Fig. Cell Survival. Doxorubicin is known for its complex pharmaco- 1a). This increase was stronger than the detected CD95L induction (Fig. logical mechanism. Therefore, we assessed the contribution of 1b) that was reported earlier (1). CD95L- and RANKL-induced apoptosis, respectively, by measuring Neutralizing RANKL Blocks Doxorubicin-induced Apoptosis. the metabolic activity over time using the MTT test (Fig. 4). Consist- We next asked if RANKL plays a nonredundant role in doxorubicin- ent with the experiments shown above, blocking CD95L or RANKL induced apoptosis in this particular cell system. The biological activity of mitigated early apoptosis-inducing cytotoxic effects of doxorubicin. secreted RANKL was neutralized by monoclonal antibodies. In the However, neither one of the antibodies was able to completely abro- presence of either one of two anti-RANKL monoclonal antibodies, doxo- gate the cytotoxicity of doxorubicin in long-term cultures. rubicin-induced apoptosis was completely inhibited for 12 h. The spec- RANKL Enhances Doxorubicin-induced NF-␬B Activation. ificity was further corroborated by a RANK-Fc fusion protein that had One of the major downstream targets that lent its name to RANKL is similar effects (Fig. 2). This experiment shows that RANKL plays an NF-␬B. To prove its involvement in the contribution of RANKL to the integral role in doxorubicin-induced apoptosis that cannot be fully com- apoptotic effect of doxorubicin, we analyzed the phosphorylation state of 1773

cytotoxic mechanism of doxorubicin leading to apoptotic cell death depends on cellular proteins because the inhibition of de novo protein synthesis almost completely abolished the apoptotic effect of these drugs (3). TNF␣-homologues and their receptors have been implicated in the cellular response to anthracycline derivatives for a number of years now (18). However, the pivotal role of CD95 in doxorubicin-induced apoptosis is challenged by other findings, namely the capability of doxorubicin to induce apoptosis in CD95L-resistant cell lines (19). The potential involvement of other TNF␣-homologues has also been investigated, most thoroughly the contribution of TRAIL. In several experiments, TRAIL was shown to be up-regulated after the addition of an anthracyclin derivative (7). It was even more interesting to learn that these mechanisms are relevant to the in vivo situation as well (20). Findings like these initiated a wide interest in apoptosis-based therapies, and there are numerous cellular proteins in apoptotic pathways that represent promising drug targets (21). As the family of known TNF␣-homologous ligands grew, we concentrated on RANKL. The transcription of RANKL mRNA is induced in the T-lymphoblastic cell line CEM in the presence of doxorubicin. In the presence of neutralizing antibody or RANK-Fc fusion protein, early doxorubicin-induced apoptosis is almost completely inhibited. Incubation with both RANKL and doxorubicin increased apoptosis levels several-fold. This observation is dependent on the apoptotic stim- ulus, because Staurosporine-induced apoptosis was not affected by the presence of neutralizing ␣RANKL antibody. Moreover, the effect might

Fig. 2. Apoptosis assays. Coincubation of CEM cells with ␣RANKL mAb clone M365 or RANK-Fc fusion protein inhibits doxorubicin-induced apoptosis. The neutralizing effect of ␣CD95L mAb served as comparison. Apoptosis was assessed by annexin V staining and exclusion of propidium iodide in flowcytometric analysis (a, three independent experiments). The trend of these results was corroborated by the assessment of internucleosomal DNA cleavage, a characteristic of late apoptosis (b, two independent experiments). Results in this and subsequent figures are given as mean and SD. p65NF-␬B by Western blot (Fig. 5a). There was an enhanced phosphorylation of NF-␬B in CEM cells by incubation in the presence of 1 ␮M doxorubicin after 3 h, which continued to increase after6hofincubation. However, the activation of NF-␬B was significantly stronger, when the cells were incubated in the presence of doxorubicin and 3 ␮g/ml recombinant human RANKL. These events preceded detectable apoptotic ␬ Fig. 3. Apoptosis assay. Recombinant human RANKL increases doxorubicin-induced changes by several hours. Inhibition of NF- B by the peptide SN50 apoptosis in CEM cells as assessed by annexin V staining after 12-h incubation (n ϭ 3). reduced the percentage of apoptotic cells by 25% (data not shown). Inhibition was not complete, most likely due to intracellular degradation of the peptide during the long incubation period. Staurosporine-induced Apoptosis Is not Sensitive to ␣RANKL. To further explore some of the mechanistic aspects, we focused on the involvement of mitochondria early in apoptosis induced by doxorubicin. Staurosporine served as a control, because it is known to disrupt the mitochondrial membrane potential as one of the first detectable cellular changes causing the release of cytochrome c. As shown in Fig. 5b, incubation of CEM cells with doxorubicin and Staurosporine, respectively, induced cytochrome c release from the mitochondria. However, neutralizing ␣RANKL antibody inhibited this effect only in the case of doxorubicin, whereas Staurosporine was still able to induce cytochrome c release within 10 h of incubation. In line with this finding, Staurospo- rine-induced apoptosis as assessed by staining CEM cells with annexin V was not affected by the presence of this antibody either (data not shown).

Discussion Fig. 4. MTT test. ␣RANKL mAb, RANK-Fc, and ␣CD95L mAb protected the metabolic activity of CEM cells of doxorubicin; however, they finally failed to completely Most cytostatic drugs, such as the anthracycline derivative doxorubi- block cytotoxicity of doxorubicin over time (n ϭ 2). Results are given as mean, and cin, aim at several cellular targets, many of which are unclear. The early untreated CEM cells served as control. 1774

are needed to evaluate the efficiency of a combined administration of doxorubicin and RANKL. Eventually, this could lead to increased cytotoxicity of anthracyclin derivatives against malignant cells and reduced side effects by allowing for dose reduction of this chemotherapeutic agent.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. Receptor Activator of Nuclear Factor κB Ligand Plays a Nonredundant Role in Doxorubicin-induced Apoptosis

Ingo Müller, Stefan M. Pfister, Ulrike Grohs, et al.

Cancer Res 2003;63:1772-1775.

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