The Protein Kinase C Activators Phorbol Esters And

Home , Acid sphingomyelinase, Phosphatidylserine

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 VÃ©roniqueMansat,Guy Laurent, Thierry Levade, All BettaIeb, and Jean-Pierre JaffrÃ©zou2 Contra! Jeune Formation, institut National de Ia Sante et de Ia Recherche MÃ©dicale 9503, Centre Claudius RÃ©gaud,Toulouse Cedex 31052 [V. M., G. L, A. B., f-P. J.J; Laboratoire de Biochimie MÃ©dicale,Institut National de Ia Sante et de Ia Recherche MÃ©dicaleU466, Centre Hospitalier Universitaire Rangueil, Toulouse 31403 (T. LI: and Service d'HÃ©matologie 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 0.5â€”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 4â€”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 FÃ©dÃ©ration 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 MÃ©dicalefellowship. 2 To whom requests for reprints should be addressed, at CJF INSERM 9503, Centre Claudius RÃ©gaud,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.

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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 â€˜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 [â€˜y-32P]ATP(3000 Ci/mmol). After 30 mm at 30Â°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 media (Fig. 1, A and B), which remained above 200% for at 00 200 least 8 h (data not shown). At several time points, cells were washed Ii 150' and both neutral and acid sphingomyelinase activity was measured. 150@ As shown in Fig. 14, treatment of U937 cells with 1.0 @tMdaunoru 100@ bicin led to a >30% increase in neutral sphingomyelinase activity, 100@ which peaked at 5 mm. However, pretreatment of cells with TPA or I- phosphatidylserine completely inhibited neutral sphingomyelinase 0 0.1 1 10 100 Concentration(@tg/ml) stimulation but had no effect on basal levels (approximately 43 pmol/h/mg of protein). Acid sphingomyelinase activity levels were 0 10 20 30 40 50 60 unchanged in the presence of daunorubicin, and PKC activators had no effect on this activity (data not shown). Similar results were Time (mm) obtained in HL-60 cells (data not shown). Fig. 1. Modulation of total cellular PKC activity by TPA and phosphatidylserine. U937 To determine whether the inhibition of daunorubicin-induced cells were treated with 100 nsi TPA (A) or 50 p@g/m1phosphatidylserine (B) for different periods of time. Insets. U937 cells were treated with different concentrations of TPA for sphingomyelinase stimulation may influence sphingomyelin hydroly 3 h (A) or phosphatidylserine for 30 mm (B). Total cellular PKC activity was measured sis and ceramide generation, we measured both sphingomyelin and by the incorporation of 32P onto myelin basic protein as described in â€˜Materials and ceramide content in U937 cells pretreated with TPA and phosphati Methods.â€•Results are means of triplicate determinations (bars. SE). dylserine, compared to untreated U937 cells. As shown in Fig. 2B, 1.0 @LMdaunorubicin triggered a sphingomyelin hydrolysis cycle within 4â€”10mm. Total percentage of sphingomyelin hydrolysis in U937 about 55% of DNA fragmentation (Table 1). Both TPA and phos cells averaged 25%. As expected, in daunorubicin-treated cells, a phatidylserine significantly inhibited (>95%) the morphological fea significant burst in intracellular ceramide generation (about 35%) was tures of daunorubicin-triggered apoptosis and completely blocked observed concurrently with sphingomyelin hydrolysis at approxi both DNA laddering (Fig. 4) and fragmentation (Table 1). Interest mately 10 mm (Fig. 2C). Similar results were observed when intra ingly, the inhibition by TPA and phosphatidylserine of daunorubicin cellular ceramide was quantitated by the diacylglycerol kinase method triggered apoptosis could be relieved partially by treating cells with 25 (data not shown). However, pretreatment with TPA or phosphatidyl @LMof cell-permeant C6-ceramide (Table 1). Although the effect of serine completely abolished both sphingomyelin hydrolysis and cer the C6-ceramide was not as potent as that of daunorubicin (17% amide generation (Fig. 2, B and C). compared to 55%, respectively, in controls) when used in complete To evaluate the effects of the inhibition of ceramide generation on media, these observations nevertheless confirmed that cell permeant daunorubicin-induced apoptosis, U937 cells, pretreated with TPA or ceramides can mediate an apoptotic effect. Higher C6-ceramide con phosphatidylserine and incubated further for 1 h with daunorubicin, centrations (50â€”100ELM)led to necrotic features (data not shown). were washed and resuspended in drug-free medium. At various time Similar results were obtained in HL-60 cells (data not shown). To points, cells were analyzed for apoptosis. Daunorubicin treatment led further demonstrate the apparent link between PKC activation and to cell shrinkage and chromatin condensation in more than 90% of inhibition of daunorubicin-induced apoptosis, dose-effect studies were cells by 6 h, as observed by fluorescence microscopy (DAPI staining; performed. As shown in Fig. 5, significant effects on both ceramide Fig. 3). Quantitative analysis revealed that daunorubicin induced generation and DNA fragmentation were observed at concentrations 5301

INHIBITION OF NEUTRAL SPHINGOMYELINASEACTIVATION

phatidylserine presented any significant effect on daunorubicin uptake or retention (data not shown). In this study, we showed that PKC activators TPA and phosphati dylserine inhibited daunorubicin-induced neutral sphingomyelinase .@2 â€” â€”

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0 5 10 15 20 25 30 35 E@â€˜ !@ Fig. 3. Inhibition by TPA and phosphatidylserine of daunorubicin-triggered apoptosis in U937 cells. Cells were preincubated in the absence (A and B) or in the presence of 100 Time (mm) flM TPA for 3 h (C and D) or 50 @g/mlphosphatidylserine for 30 rain (E and F), followed by a 60-mm incubation with (B, D, and F) or without (A, C, and E) 1.0 psi daunorubicin. Fig. 2. Inhibition by TPA and phosphatidylserine of daunorubicin-thggered activation Thesewerethenwashedandincubatedfurtherfor5 h indrug-freemedia.Morphological of neutral sphingomyelinase in U937 cells. U937 cells were preincubated in the absence alterations of chromatin were evaluated by DAPI staining and viewed at a magnification (0) or the presence of 100nsi TPA for 3 h (/@)or50 @zg/m1phosphatidylserinefor 30 rain of X50. (A), followed by 1.0 p.Mdaunorubicin for the time intervals indicated. A, inhibition of neutral sphingomyelinase activation in U937 cells treated with daunorubicin. Neutral sphingomyelinase activity was determined in U937 cells as described in â€œMaterialsand Table 1 Effect of PKC activators on DNAfragmentation triggered by DNR in Methods.â€•Resultsare expressed as the percentage of sphingomyelinase activity in control U937 cellsâ€• (43 pmol/h/mg of protein). B, inhibition of sphingomyelin hydrolysis in U937 cells treated with daunorubicin. Sphingomyelin levels were estimated in U937 cells prelabeled with fragmentation [3H]choline for 48 h. Following incubation, aliquots were collected, and lipids were (%)Noneâ€”PKC activatorDNR 1 @LMC6-ceramide25 @MDNA extracted. Labeled sphingomyelin was quantitated as described in â€œMaterialsandMeth Â±0.4 ads.â€•C, inhibition of ceramide generation in U937 cells treated with daunorubicin. â€” Ceramide levels were estimated in U937 cells prelabeled with [9,lO-3Hjpalmitic acid for + 17.0Â±4.0 +â€” â€”4.3 48 h. Following incubation, aliquots were collected, and lipids were extracted. Labeled 55.6Â±6.4TPAâ€”

ceramide was resolved by analytical TLC as described in â€œMaterialsandMethods.â€•Inall â€” cases, results are representative of at least three independent experiments (data are means; + 5.4Â±1.3 â€” + 14.7Â±2.8 bars, SE). SEs for TPA and phosphatidylserine are <10% and were omitted for clarity. 15.3Â±2.5Phosphatidylserineâ€”+â€” +4.9Â±0.7 *, P <0.01. Â±0.4 + â€” 5.5Â±1.5 â€” + 13.4Â±2.9 +â€” +4.6 14.0Â±3.1 that stimulated PKC activity by at least 150%. Moreover, complete a U937 cells were preincubated with or without 100 nM TPA for 3 hours or 50 pg/mI inhibition was seen with 250% PKC activation. These results further phosphatidylserine for 30 rain. Cells were then incubated with or without 1.0 @LMDNRfor showed a close dose-effect correlation of TPA and phosphatidylserine 60 mm, washed, and resuspended in drug-free medium with or without 25 @MC6- ceramide for 5 h, after which quantitative DNA fragmentation was determined by the on PKC activation and inhibition of daunorubicin-triggered ceramide spectrofluometric DAPI procedure (see â€œMaterialsand methods.)â€•The data are generation and DNA fragmentation. Finally, neither TPA nor phos means Â±SE of four independent determinations. 5302

stimulation, sphingomyelin hydrolysis, ceramide generation, and ap optosis. Only the classic (a, (3, and â€˜y)andnovel (& â‚¬,0,and @)PKC isoforms present a phorbol ester-binding site, whereas all of the PKCs have a NH2-terminal regulatory domain containing a phosphatidyl senne-binding site (for a review see Ref. 23). Therefore, our study presents evidence that daunorubicin-induced neutral sphingomyeli EC nase stimulation can be manipulated negatively by one or several tao classic and/or novel PKC isoforms but likely not by atypical (@,A, and cc 0@s-,@0 i.) isoforms activatable by lipid cofactors, such as phosphatidylinosi = tol-3 phosphate and ceramide itself (24â€”27). The mechanism by which PKC regulates sphingomyelinase activity remains to be elucidated. The fact that PKC activation had no influ ence on basal sphingomyelin activity suggests that PKC regulates signaling events triggered by daunorubicin upstream of sphingomy elinase stimulation. Recently, it has been suggested that proteolytic C events precede daunorubicin-induced sphingomyelinase stimulation 0 50@ and ceramide generation (28, 29). Therefore, it can be speculated that @U) sphingomyelinase activation is controlled by both phosphorylation and proteolytic processes; e.g., it could be possible that sphingomy elinase is recruited to the plasma membrane after phosphorylation and Eo 0)0 proteolytic processing or degradation of an inhibitory protein (30). cc @ Is Alternatively, it could be possible that phosphorylation is needed to activate a critical protease responsible for the cleavage of a proform 4 into an active form of the sphingomyelinase. z ,@ 10- Regardless of the mechanism by which PKC regulates sphingomy elinase stimulation, our fmdings may have important implications in anthracycline pharmacology. Indeed, a number of intrinsic and envi 0 0.1 1 10 100 ronmental factors strongly influence PKC activity and, therefore, may Concentration contribute to resistance to these drugs. For example, cytokines and growth factors, such as tumor necrosis factor a, interleukin 3, or (nMEi, @tg/mIA) granulocyte-macrophage colony-stimulating factor, induce the diacyl Fig. 5. Dose effect of TPA and phosphatidylserine on daunorubicin-triggered ceramide glycerol formation through hydrolysis of phosphatidylcholine (31â€” generation and DNA fragmentation. U937 cells were treated with different concentrations of TPA for 3 h (L@.)orphosphatidylserine for 30 mm (A), followed by a 60-mm incubation 34). Furthermore, oncogenes, such as c-Abl variants, may interact with or without 1.0 p@Mdaunorubicin. A, ceramide levels were estimated in U937 cells directly with phospholipase C and contribute to accelerated diacylg prelabeled with [9,lO-3Hlpalmitic acid for 48 h. Following incubation, aliquots were lycerol turnover (35, 36). Because diacylglycerol activates PKC, it is collected, and lipids were extracted. Labeled ceramide was resolved by analytical TLC as described in â€œMaterialsandMethods.â€•Resultsrepresent peak ceramide generation oh conceivable that paracrine or autocrine production of cytokines, as served at 4â€”10mm.(B), quantitative DNA fragmentation was determined by the spec trofluorometric DAPI procedure as described in â€œMaterialsand Methods.â€•Results are means of triplicate determinations (bars, SE). z c@ @ z well as abnormal oncogene expressions that may lead to constitutive @@@ p.- ,4@+ Ã· phospholipase C activation, may limit sphingomyelinase stimulation, ceramide generation, and apoptosis in anthracycline-treated cells. One I could speculate that such a mechanism could account for the lack of apoptotic response to daunorubicin observed in certain leukemia cells (2). Finally, PKC emerges as a critical negative regulator of anthracy dine-activated sphingomyelin-ceramide apoptotic pathway. PKC may potentially act through different mechanisms: inhibition of the apop totic effect of ceramide (37); stimulation of sphingosine kinase activ ity (38), resulting in accumulation of sphingosine-l-phosphate, a potent apoptosis inhibitor (39); inhibition of ceramide production (as in this study); and perhaps other mechanisms. These findings may explain the results of previous studies that showed a correlation between PKC activity and clinical resistance to anthracyclines (1), as well as the induction of resistance to doxorubicin by phorbol esters (40).

REFERENCESgeredFig. 4. Inhibition by TPA and phosphatidylserine (PS) of daunorubicin (DNR)-trig presenceofDNA laddering in U937 cells. Cells were preincubated in the absence or In:incubation100 nsaTPA for 3 h or 50 @zWmiphosphatidylserinefor 30 mm, followed by a 60-mm1. Chabner. B. A., and Myers, C. E. Clinical pharmacology of cancer chemotherapy. andfurther with or without 1.0 psi daunorubicin. These were then washed and incubatedV. T. J. DeVitta, S. HeIlman, and S. A. Rosenberg (cdx.), Cancer: Principles 1989.detenninedfor S h in drug-free media. The formation of oligonucleosomal fragments wasPractice of Oncology, pp. 349â€”395.Philadelphia: J. B. Lippincoti Co.. Bailly,are bylysingcellsandsubmittingtheDNAtoagarosegelelectrophoresis.Results2. Quillet-Mary, A., Mansat, V., Duchayne, E., Come, M. G., Allouche, M., representative of at least three independent experiments.J. D., Bordier, C., and Laurent, G. Daunorubicin-induced internucleosomal DNA 5303

fragmentation in acute myeloid cell lines. Leukemia (Baltimore), 10: 417â€”425, dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. 1996. Cell, 78: 1005â€”1008,1994. 3. Bailly, J. D., Skladanowski, A., Bettaleb, A., Mansat, V., Larsen, A. K., and Laurent, 22. Kitano, T., Go, M., Hikkawa, U., and Nishizuka, Y. Assay and purification of protein G. Natural resistance of acute myeloid leukemia cell lines to mitoxantrone is asso kinase C. Methods Enzymol., 124: 349â€”352,1986. ciated with lack of apoptosis. Leukemia (Baltimore), 11: 1523â€”1532,1997. 23. Nishizuka, Y. Protein kinase C and lipid signaling for sustained cellular responses. 4. Hannun, Y. A. Apoptosis and the dilemma of cancer chemotherapy. Blood, 89: FASEB J.. 9: 484-496, 1995. @ 1845â€”1853,1997. 24. Nakanishi, H., and Exton, J. H. Purification and characterization of the isoform of 5. JaffrÃ©zou,J. P., Levade, T., Bettaleb, A., Andrieu, N., Bezombes, C., Macsire, N., protein kinase C from bovine kidney. J. Biol. Chem., 267: 16247â€”16354, 1992. @ Vermeersch, S., Rousse, A., and Laurent, G. Daunorubicin-induced apoptosis: trig 25. Nakanishi, H., Brewer, K. A., and Exton, J. H. Activation of the isoform of protein gering of ceramide generation through sphingomyelin hydrolysis. EMBO J., 15: kinaseC by phosphatidylinositol3,4,5-trisphosphate.J.Biol.Chem.,268: 13-16, 2417â€”2424, 1996. 1993. 6. Hannun, Y. A. Functions of ceramide in coordinating cellular responses to stress. 26. Lozano, J., Berra, E., Municio, M. M., Diaz-Meco, M. T., Dominguez, I., Sanz, L., @ Science (Washington DC), 274: 1855â€”1861,1996. and Moscat, J. Protein kinase C isoform is critical for KB-dependent promoter 7. Allouche, M., Bettaleb, A., Vindis, C., Rousse, A., Grignon, C., and Laurent, G. activation by sphingomyelinase. J. Biol. Chem., 269: 19200-19202, 1994. Influence of Bcl-2 overexpression on the ceramide pathway in daunorubicin-induced 27. Muller, G., Ayoub, M., Storz, P., Rennecke, J., Fabbro, D., and Pfizenmaier, K. PKC apoptosis of leukemic cells. Oncogene, 14: 1837â€”1845,1997. Cisa molecularswitchinsignaltransductionof1'NFa,bifunctionallyregulatedby 8. Jarvis, W. D., Fornari, F. A., Traylor, R. S., Martin, H. A., Kramer, L. B., Erukulla, ceramide and arachidonic acid. EMBO J., 14: 1961â€”1969,1995. R. K., Bittman, R., and Grant, S. Induction of apoptosis and potentiation of ceramide 28. Dbaido, G. S., Perry, D. K., Gamard, C. J., Plan, R., Poirier, G. G., Obeid, L. M., and mediated cytotoxicity by sphingoid bases in human myeloid leukemia cells. J. Biol. Hannun, Y. A. Cytokine response modifier A (Cnn A) inhibits ceramide formation in Chem., 271: 8275â€”8284,1996. response to tumor necrosis factor (TNF)-a: Crm A and Bcl-2 target distinct compo 9. Jarvis, W. D., Turner, A. J., Povirk, L. F., Traylor, R. S., and Grant, S. Induction of nents in the apoptotic pathway. J. Exp. Med., 185: 481â€”490,1997. apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leuke 29. Mansat, V., Bettaleb, A., Levade, T., Laurent, G., and JaffrÃ©zou,J. P. Serine protease mia cells by pharmacological inhibitors of protein kinase C. Cancer Res., 54: inhibitors block neutral sphingomyelinase activation, ceramide generation, and ap 1707â€”1714,1994. optosis triggered by daunorubicin. FASEB J., 11: 695â€”702,1997. 10. Chmura, S. J., Nodzenski, E., Weichselbaum, R. R., and Quintans, J. Protein kinase 30. Bezombes, C., Maestre, N., Laurent, G., Levade, T., Benaleb, A., and Jaffr@zou, J. P. C inhibition induces apoptosis and ceramide production through activation of a Restoration of TNFa-induced ceramide generation and apoptosis in resistant human neutral sphingomyelinase. Cancer Rca., 56: 2711â€”2714, 1996. leukemic KG1a cells by the P-glycoprotein blocker PSC833. FASEB J., in press, 11. Haimovitz-Friedman, A., Kan, C. C., Ehleiter, D., Persaud, R. S., McLoughlin, M., 1997. Fuks, Z., and Kolesnick, R. N. Ionizing radiation acts on cellular membranes to 31. Schfitze, S., Berkovic, D., Tomsing, 0., Unger, C., and Kronke, M. Tumor necrosis generate ceramide and initiate apoptosis. J. Exp. Med., 180: 525â€”535,1994. factor induces rapid production of 1,2-diacylglycerol by a phosphatidylcholine 12. Escargueil-Blanc, I., Salvayre, R., and NÃ¨gre-Salvayre, A. Necrosis and apoptosis specific phospholipase C. J. Exp. Med., 174: 975â€”988,1991. induced by oxidized low density lipoproteins occur through two calcium-dependent 32. Duronio, V., Nip, L., and Pelch, S. L. Interleukin 3 stimulates phosphatidylcholine pathways in lymphoblastoid cells. FASEB J., 8: 1075â€”1080,1994. turn over in a mast-megacaryocyte cell line. Biochem. Biophys. Res. Commun., 164: 13. Gong, J., Traganos, F., and Darzynkiewicz, Z. A selective procedure for DNA 804â€”807, 1989. extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. 33. Rao, P., and Mufson, R. A. Human interleukin-3 induces activation of phosphatidyl Anal. Biochem., 218: 314â€”319, 1994. choline-specific phospholipase C and translocation of protein kinase C. Cancer Res., 14. McConkey, D. J., Aguilar-Santelises, M., Harztell, P., Eriksson, I., Mellstedt, H., 54: 777â€”781,1994. Orrenius, S., and Jondal, M. Inhibition of DNA fragmentation in thymocytes and 34. Rao, P., and Mufson, R. A. Human interleukin-3 rapid induction of phosphati isolated thymocyte nuclei by agents that stimulate protein kinase C. J. Immunol., 146: dylcholine hydrolysis is independent of protein kinase C but dependent on 1072â€”1077,1989. tyrosine phosphorylation in transfected NIH 3T3 cells. J. Immunol., 154: 1664â€” 15. Kapuscinski. J., and Skooczylas, B. Simple and rapid fluorometric method for DNA 1670, 1995. microassay. Anal. Biochem., 83: 252â€”257,1977. 35. Gotoh, A., Miyazawa, K., Ohyashiki, K., and Toyama, K. Potential molecules 16. Andrieu, N., Salvayre, R., and Levade, T. Evidence against involvement of the acid implicated in downstream of p185BcR.ABL in Ph@' ALL involve G1'Pase-activating lysosomal sphingomyelinase in the tumor-necrosis-factor- and interleukin-I-induced protein, phospholipase C-'yl, and phosphatidylinositol 3'-kinase. Leukemia (Balti sphingomyelin cycle and cell proliferation in human fibroblasts. Biochem. J., 303: more), 8: 115â€”120,1994. 341â€”345,1994. 36. Owen, P. J., Musk, P., Evans, C. A., and Whetton, A. D. Cellular signaling events 17. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, elicited by v-abl associated with growth factor independence in a interleukin-3- M. D., Fujimoto,E K., Goeke, N. M., Olson, B. J., and Klenk, D. C. Measurementof dependent cell line. J. Biol. Chem., 268: 15696â€”15703, 1993. proteins using bicinchoninic acid. Anal. Biochem., 150: 76â€”85,1985. 37. Jarvis, W. D. J., Fornari, F. A., Browning, 3. L., Gewirtz, D. A., Kolesnick, R. N., and 18. Folch, J., Lees, M., and Sloane-Stanley, G. H. A simple method for the isolation and Grant, S. G. Attenuation of ceramide-induced apoptosis by diglyceride in human purification of total lipids from animal tissues. J. Biol. Chem., 226: 497â€”509,1957. myeloid leukemia cells. J. Biol. Chem., 269: 31685â€”31692,1994. 19. Stoffel, W., and Melzner, I. Studies in vitro on the biosynthesis of ceramide and 38. Spiegel, S., and Milstien, S. Sphingoid bases and phospholipase D activation. Chem. sphingomyelin: a reevaluation of proposed pathways. Hoppe-Seyler's Z. Physiol. Phys. Lipids, 80: 27â€”36,1995. Chem., 361: 755â€”771,1980. 39. Cuvillier, 0., Pirianov, G., Kleuser, B., Vanek, P. G., Coso, 0. A., Gutkind, J. S., and 20. Levade, T., Tempesta, M. C., and Salvayre, R. The in situ degradation of ceramide, Spiegel, S. Suppression ofceramide-mediated programmed cell death by sphingosine a potential lipid mediator, is not completely impaired in Farber disease. FEBS Lett., 1-phosphate. Nature (Lond.), 381: 800â€”803, 1996. 329: 306â€”312,1993. 40. Fine, R. L., Patel, J., and Chabner, B. Phorbol esters induce multidrug resistance in 21. Wiegmann, K., Schtitze, S. Machleidt, T, Witte, D., and Kronke, M. Functional human breast cancer cells. Proc. Nati. Aced. Sci. USA, 85: 582â€”586,1988.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1997 American Association for Cancer Research. The Protein Kinase C Activators Phorbol Esters and Phosphatidylserine Inhibit Neutral Sphingomyelinase Activation, Ceramide Generation, and Apoptosis Triggered by Daunorubicin

Véronique Mansat, Guy Laurent, Thierry Levade, et al.

Cancer Res 1997;57:5300-5304.

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