ICANCER RESEARCH57. 5300-5304. December 1, 19971 The Protein Kinase C Activators Phorbol Esters and Phosphatidylserine Inhibit Neutral Sphingomyelinase Activation, Ceramide Generation, and Apoptosis Triggered by Daunorubicin1 VéroniqueMansat,Guy Laurent, Thierry Levade, All BettaIeb, and Jean-Pierre Jaffrézou2 Contra! Jeune Formation, institut National de Ia Sante et de Ia Recherche Médicale 9503, Centre Claudius Régaud,Toulouse Cedex 31052 [V. M., G. L, A. B., f-P. J.J; Laboratoire de Biochimie Médicale,Institut National de Ia Sante et de Ia Recherche MédicaleU466, Centre Hospitalier Universitaire Rangueil, Toulouse 31403 (T. LI: and Service d'Hématologie Centre Hospitalier Universitaire Purpan. Toulouse 31059 (G. LI, France ABSTRACT vincristine, ionizing radiation, anti-Fas, and tumor necrosis factor a-induced apoptosis (for a review, see Ref. 6). To address the role of protein kinase C (PKC) in the regulation of The sphingomyelin-cerarnide pathway appears to be efficiently ceramide production, we evaluated the impact of the PKC activators regulated downstream of ceramide generation. For example, we re 12.O-tetradecanoylphorbol-13-acetate and phosphatidylserine on the ap. ported recently that Bcl-2 overexpression blocked both daunorubicin optotic signaling pathway triggered by the chemotherapeutic drug dauno rubicin. Treatment of U937 and HL-60 cells with 0.5—1 @aMdaunorubicin and cell-permeant ceramide-induced apoptosis of leukemic cells but induced a greater than 30% activation of neutral sphingomyelinase activ did not affect daunorubicin-induced ceramide generation (7). In pre ity within 4—10 mm with concomitant sphingomyelin hydrolysis and vious studies, Jarvis et a!. (8) showed that not only PKC3 activators ceramide generation. Activation of PKC by 12-O-tetradecanoylphorbol including phorbol esters (TPA) and diacylglycerol inhibited the ability 13-acetate and phosphatidylserine inhibited daunorubicin-induced neu of cell-permeant ceramides to induce apoptosis but also that PKC tral sphingomyelinase activation, sphingomyelin hydrolysis, ceramide inhibitors enhanced ceramide-induced apoptosis (9). From these stud generation, and apoptosis. The apoptotic response could be restored by ies, it has been suggested that PKC is a key negative regulator of the addition of 25 ,.aMcell-permeant C6-ceramide. In conclusion, PKC ceramide-induced apoptosis. emerges as a potentially critical negative regulator of the anthracycline Little is known about the role of PKC in the regulation of ceramide activated sphingomyelin-ceramide apoptotic pathway. production. It has been reported recently that PKC inhibitors triggered neutral sphingomyelinase, suggesting that PKC may also play an INTRODUCTION important role by regulating basal sphingomyelinase activity (10). Anthracyclines are one of the most active antitumor compounds These findings are consistent with those reported by Haimovitz used in clinical oncology, especially in the treatment of acute leuke Friedmann et a!. (11), who observed that TPA inhibited ceramide mias. Despite intense efforts, their mechanism of action is still not generation and apoptosis in irradiated bovine endothelial cells. fully understood. Indeed, it has been documented that anthracyclines In this study, we present evidence that daunorubicin-triggered induce membrane alterations through lipid peroxidation; generate sphingomyelinase activity, ceramide generation, and apoptosis in my eloid leukemia cells can be inhibited by the PKC activators TPA and radical oxygen species due to electron transfer from the semiquinone ring; disturb mitochondrial homeostasis; intercalate into nuclear phosphatidylserine. DNA; and interact with both topoisomerase I and II, leading to covalent binding of these enzymes to DNA and subsequent DNA MATERIALS AND METHODS breaks (for a review, see Ref. 1). More recently, it has been shown that anthracyclines induce apoptosis in some, but not all, cellular models Drugs and Reagents. Daunorubicin was obtained from the National Can (2, 3). Present knowledge does not allow us to determine whether cer Institute Drug Repository. TPA and bovine brain phosphatidylserine were apoptosis simply reflects membrane and/or DNA lesions or represents from Sigma Chemical Co. (Paris, France). All drugs were diluted in ethanol an independent cytotoxic mechanism triggered by a specific signaling (final dilution, <0.01%). pathway (for a review, see Ref. 4). Cell Culture. The humanmonocyticleukemiacell lines U937 andHL-60, In a recent report, we demonstrated that daunorubicin activates the purchased from the American Type Culture Collection (Rockville, MD), were sphingomyelin-ceramide cycle. Indeed, daunorubicin stimulated neu cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mist glutamine, 100 units/ml penicillin, and 100 p@g/ml streptomycin (all from tral sphingomyelinase activity responsible for sphingomyelin hydrol Eurobio, Les Ulis, France). Cell stocks were screened routinely for Myco ysis and subsequent ceramide generation in U937 and HL-60 human plasma (Stratagene Mycoplasma PCR kit; Stratagene, La Jolla, CA). leukemia cells (5). The fact that cell-permeant ceramides, as well as Cytochemical Staining. Changes in cellular nuclear chromatin were eval natural ceramide (generated by exposure of the cells to bacterial uated by fluorescence microscopy by DAPI staining (12). sphingomyelinase), induce apoptosis in these cells suggests strongly DNA Analyses. DNA was resolved on a 1.8%agarosegel and visualized that ceramide was the mediator of daunorubicin-induced apoptosis. with ethidium bromide as described previously (13). Quantitative DNA frag Such an apoptotic signaling pathway has also been described in mentation was determined by the spectrofluorometric DAPI procedure as described previously (14, 15). Metabolic Cell Labeling and Sphingolipid Quantitation. Sphingomyelin Received 5/1/97; accepted 10/3/97. The costs of publication of this article were defrayed in part by the payment of page quantitation was performed by labeling cells to isotopic equilibrium with 0.4 charges. This article must therefore be hereby marked advertisement in accordance with @Ci/mlof[methyl-3H]choline(specific activity 81.0 Ci/mmol, DuPont-NEN, 18 U.S.C. Section 1734 solely to indicate this fact. Les Ulis, France) for 48 h in complete medium as described previously (5, 16). I This study was supported in part by Grant 960031 15 from La Fédération Nationale Cells were then washed and resuspended in serum-free medium for kinetic des Centres de Lutte Contre Ic Cancer (to J-P. J. and T. L.); by a grant from the Conseil Regional Midi-Pyrbes (to T. L. and J-P. J.); by l'Association pour Ia Recherche sur Ic experiments. Aliquots were taken for protein determination (17). Radioactive Cancer Grants 6749 (to G. L.), 3002 (to T. L.), and 2069 (to J-P. J.); and by a grant from lipids were extracted (1 8), and sphingomyelin was quantitated by scintillation La Ligue Nationale Contre Ic Cancer (to G. L.). V. M. is the recipient of an Institut counting (16, 19). National de Ia Sante Ct de Ia Recherche Médicalefellowship. 2 To whom requests for reprints should be addressed, at CJF INSERM 9503, Centre Claudius Régaud,20 rue du Pont St. Pierre, Toulouse Cedex 31052 France. E-mail: 3 The abbreviations used are: PKC, protein kinase C; TPA, l2-O-tetradecanoylphor [email protected]. bol-13-acetate; DAPI, 4',6'-diamidino-2-phenylindole. 5300 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1997 American Association for Cancer Research. INHIBITION OF NEUTRAL SPHINGOMYELINASE ACTIVATION Total cellular ceramide quantitation was performed by labeling cells to isotopic equilibrium with I @CiJmlof [9,lO-3H]palmitic acid (53.0 Cilmmol, Amersham, Les Ulis, France) for 48 h in complete medium as described previously (5). Cells were then washed and resuspended in serum-free medium for kinetic experiments. Lipids were extracted and resolved by TLC (20), and >1 ceramide was scraped and quantitated by liquid scintillation spectrometry. .; =. 300 Statistical analyses were performed by Student's r test. Neutral and Acid Sphingomyelinase Assay. Sphingomyelinase activities were determined as described previously (21) using [choline-methyl 0 00 ‘4C]sphingomyelin(54.5mCilmmol; DuPont-NEN; 120,000 dpm/assay) as substrate (5). PKC Assay. Total PKC activity was determinedby measuringthe incor @ poration of 32P into myelin basic protein (22). Briefly, 5 X 106 cells were lysed 150 in 20 mM Tris-HC1 (pH 7.4), 60 mM glycerophosphoric acid, 10 mM EGTA, 20 mM MgCl2, 0.1 mrvisodium fluoride, 2 ii@MDli', 1 mrst sodium orthovanadate, 20 @sg/mlaprotinin, and 1 mM phenylmethylsulfonyl fluoride. After 15 mm, the assay was initiated by the addition of 0.1 mtvt ATP, 2.5 mg/ml myelin basic protein, and [‘y-32P]ATP(3000 Ci/mmol). After 30 mm at 30°C,the reaction was stopped by the addition of 10% trichloroacetic acid and spotted on 0 1 2 3 4 5 6 phosphocellulose discs. Following extensive washes with 10% tricholoacetic acid, water, and finally ethanol, the incorporation of 32P into myelin basic Time (hr) protein was measured by scintillation counting. RESULTS AND DISCUSSION To ascertain the potential effects of PKC activators on daunorubi 250 >1 cm-induced neutral sphingomyelinase stimulation, U937 cells were @ preincubated with 100 nMTPA for 3 h or 50 @.tg/mlphosphatidylserine > for 30 mm, after which daunorubicin was added. These were consid 200 ered as optimal conditions for PKC activation of U937 cells in 0 250 complete