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Cell ApoptosisCeramide Mediates Tumor-Induced Dendritic

Lotze and Andrew A. AmoscatoTatsuya Kanto, Pawel Kalinski, Oriana C. Hunter, Michael T.

http://www.jimmunol.org/content/167/7/3773doi: 10.4049/jimmunol.167.7.3773

2001; 167:3773-3784; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/167/7/3773.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Ceramide Mediates Tumor-Induced Dendritic Cell Apoptosis1

Tatsuya Kanto,*‡ Pawel Kalinski,*‡ Oriana C. Hunter,* § Michael T. Lotze,*‡ andAndrew A. Amoscato2†‡§

Induction of apoptosis in dendritic cells (DC) is one of the escape mechanisms of tumor cells from the immune surveillance system.This study aimed to clarify the underlying mechanisms of tumor-induced DC apoptosis. The supernatants (SN) of murine tumorcell lines B16 (melanoma), MCA207, and MCA102 (fibrosarcoma) increased C16 and C24 ceramide as determined by electrospraymass spectrometry and induced apoptosis in bone marrow-derived DC.N-oleoylethanolamine or D-L-threo 1-phenyl-2-de-canoylamino-3-morpholino-1-propanol (PDMP), which inhibits acid ceramidase or glucosylceramide synthase and then increasesendogenous ceramide, enhanced DC apoptosis and ceramide levels in the presence of tumor SN. Pretreatment withL-cycloserine,an inhibitor of de novo ceramide synthesis, or phorbol ester, 12-O-tetradecanoylphorbol-13-acetate reduced endogenous ceramidelevels and protected DC from tumor-induced apoptosis. However, other DC survival factors, including LPS and TNF-�, failed todo so. The protective activity of 12-O-tetradecanoylphorbol-13-acetate is abrogated by pretreatment with phosphoinositide 3-ki-nase (PI3K) inhibitor, LY294002. Therefore, down-regulation of PI3K is the major facet of tumor-induced DC apoptosis. TumorSN, N-oleoylethanolamine, or PDMP suppressed Akt, NF-�B, and bcl-xL in DC, suggesting that the accumulation of ceramideimpedes PI3K-mediated survival signals. Taken together, ceramide mediates tumor-induced DC apoptosis by down-regulation ofthe PI3K pathway. The Journal of Immunology, 2001, 167: 3773–3784.

T umors are well known sources of a variety of biologicalsubstances. To create a favorable environment for theirenhanced growth, they release certain immunosuppres-

sive factors to evade the immune surveillance system of the host.Dendritic cells (DC)3 are arguably the most potent APC in vivoboth in priming naive T cells and maintaining specific immuneresponses against tumors (1). From the analyses of tumor speci-mens obtained from patients, the reduced number of DC infiltratedinto tumor correlates with a poor prognosis for patients (2, 3).These observations suggested that DC residing in the tumor mi-croenvironment generally play a beneficial role for patients withregard to anti-tumor immune reactions. However, the phenotypesor functions of DC could be altered in the presence of tumor. DCinfiltrated in tumor tissue or recovered from the peripheral blood ofpatients showed reduced expression of costimulatory molecules

and defective cytokine production (4, 5), implying that tumor-de-rived factors impede DC maturation.

To achieve a better T cell response against tumors, the presenceof functionally active APC or DC in T cell areas may be required.Additionally, the longevity of DC in vivo also contributes to ob-taining a favorable T cell response (6), suggesting that DC primedtoward apoptosis are unable to support T cell reactions. The in-crease of apoptosis in DC coincubated with tumor cells and thepaucity of DC in implanted tumors have been reported both inhuman and mouse systems (7, 8). These findings postulated theexistence of tumor-derived apoptotic factors; however, such fac-tors have yet to be characterized or identified. Although the pre-treatment of DC with IL-12 or CD40 ligand (7) or the transductionof bcl-xL (8) is efficacious in rescuing DC from tumor-inducedapoptosis to some extent, the underlying mechanisms involved inincreasing DC susceptibility to apoptosis remain unsolved.

DC apoptosis is a physiological phenomenon involved both inthe development of DC from precursors and in the elimination ofend-stage mature cells. The susceptibility of DC to apoptosis isregulated in the course of their development (9). The signal trans-duction pathway of DC apoptosis or survival must be fully ex-plored to modulate DC survival.

Cumulative reports have focused on the importance of cer-amides as bioeffector molecules involved in cellular stress re-sponses as well as in programmed cell death (10). Intracellularceramide interacts with several signaling pathways to transducesignals and determine cell fate (10). Nevertheless, the relevance ofceramide for apoptosis is still controversial (11), which is mainlydue to the differences in the procedures of ceramide quantification,types of cells used, or the stimuli used to induce apoptosis. Cur-rently, little is known about the implication of ceramide in DCbiology, except for the positive impact on DC maturation as as-sessed by the decrease of phagocytic activity (12).

In this study, we investigated the mechanisms of tumor-inducedDC dysfunction or apoptosis. We found that several tumor super-natants (SN) increased ceramide and induced apoptosis in bone

*Department of Surgery, Division of Biologic Therapeutics and Surgical Oncology,†Department of Pathology, and ‡University of Pittsburgh Cancer Institute, Pittsburgh,PA 15261; and §University of Pittsburgh Mass Spectrometry Facility, University ofPittsburgh Center for Biotechnology and Bioengineering, Pittsburgh, PA 15219

Received for publication March 20, 2001. Accepted for publication August 1, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by National Institutes of Health Grants1RO1CA82016-01A29, 5P30CA47904-12, and IPO1CA73743-04.2 Address correspondence and reprint requests to Dr. Andrew A. Amoscato, Depart-ment of Surgery, University of Pittsburgh, Center for Biotechnology and Bioengi-neering, 300 Technology Drive, Suite 206, Pittsburgh, PA 15219. E-mail address:amoscatoaa@msx.upmc.edu3 Abbreviations used in this paper: DC, dendritic cell; Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-CHO; BM, bone marrow; C2 ceramide, N-acetyl-D-erythro-sphin-gosine; C2 dihydroceramide, N-dihydroacetyl-D-erythro-sphingosine; ERK, extracel-lular signal-related kinase; ESI-MS, electrospray ionization mass spectrometry; FB1,fumonisin B1; m/z, mass to charge ratio; NAO, 10-nonylacridine orange; NOE, N-oleoylethanolamine; PC, phosphatidylcholine; PDMP, D-L-threo 1-phenyl-2-de-canoylamino-3-morpholino-1-propanol; PI, propidium iodide; PI3K, phosphoinosi-tide 3-kinase; PKC, protein kinase C; Rh123, rhodamine 123; SAC, Staphylococcusaureus Cowan I; SAPK, stress-activated protein kinase; SM sphingomyelin; SMase,sphingomyelinase; SN, supernatant; TIC, total ion current; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; Z-VAD-fmk, Z-Val-Ala-Asp (Ome)-fluorometylketone; PIn,phosphatidylinositol; FasL, Fas ligand.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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marrow (BM)-derived DC. We demonstrate that ceramide medi-ates tumor-induced DC apoptosis through the down-regulation ofphosphoinositide 3-kinase (PI3K) and its downstream signals, Akt,NF-�B, or bcl-xL. DC survival factors, including LPS or TNF-�,failed to prevent tumor-induced DC apoptosis. However, the re-duction of ceramide was effective for protecting DC from apopto-sis and promoting DC survival in the presence of tumor.

Materials and MethodsMice

Three- to 5-wk-old female C57BL/6 mice (H-2Kb, I-Ab) and BALB/c mice(H-2Kd, I-Ad) were purchased from The Jackson Laboratory (BarHarbor, ME).

Reagents

Murine GM-CSF and IL-4 were provided by Schering-Plough (Kenilworth,NJ). Cell-permeable inhibitors specific for caspase-3 (N-acetyl-Asp-Glu-Val-Asp-CHO, Ac-DEVD-CHO), were obtained from BD PharMingen(San Diego, CA) and caspase-1 inhibitor, Z-Val-Ala-Asp (OMe)-fluorome-tylketone (Z-VAD-fmk), from Alexis Biochemicals (San Diego, CA),respectively. Staphylococcus aureus Cowan I (SAC), N-acetyl-D-erythro-sphingosine (C2 ceramide), fumonisin B1 (FB1), L-cycloserine, and12-O-tetradecanoylphorbol-13-acetate (TPA) were obtained from Sigma(St. Louis, MO). N-dihydroacetyl-D-erythro-sphingosine (C2 dihydrocer-amide), glucosylceramide synthase inhibitor, D-L-threo 1-phenyl-2-decan-oylamino-3-morpholino-1-propanol (PDMP), and protein kinase C (PKC)inhibitor, bisindoylmaleiamide I were purchased from Calbiochem (LaJolla, CA). Inhibitors of extracellular signal-related kinase (ERK)(PD98059) and p38 stress-activated protein kinase (SAPK) (SB20358)were obtained from Alexis Biochemicals. PI3K inhibitor (LY294002) andacid ceramidase inhibitor (N-oleoylethanolamine, NOE) were obtainedfrom Biomol (Plymouth Meeting, PA).

Cell lines and culture SN

Cell lines syngeneic to C57BL/6 were used to collect culture SN; B16, BL6(melanoma), MC38 (adenocarcinoma), MCA205, MCA102, and MCA207(fibrosarcoma). Allogeneic cell lines, SCC-VII (squamous cell carcinoma)and TBJ (neuroblastoma), were also used. As controls for these tumor celllines, we used the mouse melanocyte cell line (Melan-A) and the mousefibroblast cell line (L cells). Melan-A was provided from Dr. E. Gorelik(University of Pittsburgh, Pittsburgh, PA) and L cells were purchased fromAmerican Type Culture Collection (Manassas, VA), respectively. All cellswere maintained in complete medium composed of RPMI 1640 (Life Sci-ence Technologies, Gaithersburg, MD) supplemented with 10% FBS, 2mM L-glutamine, 1% nonessential amino acids, and 100 U/ml penicillinand streptomycin. To prepare the culture SN, 1–2 � 106 cells were seededin 20 ml of complete medium on 165-cm2 flasks and cultured for 3 or 4days. Before reaching confluence, SN were collected and centrifuged at1500 � g for 5 min to remove residual dead cells. All SN were stored at�80°C until use.

Generation of BM DC

BM DC were generated according to the method described previously (7),with some modification. In brief, BM cells were depleted of CD4�, CD8�,or B220-positive cells with the treatment of anti-mouse CD4�, CD8�, andB220 Abs (recovered from hybridoma cell lines RL174, TIB145, andTIB146, respectively) and rabbit complement. Nonadherent cells were cul-tured in complete medium containing 1000 U/ml each GM-CSF and IL-4at 37°C, 5% CO2. On day 7 of culture, CD11c-positive cells were separatedusing anti-mouse CD11c Ab-bound magnetic beads (Miltenyi Biotec, Au-burn, CA) according to the manufacturer’s instructions. The cells weregiven 20–50% (v/v) cellular SN and were cultured thereafter.

Flow cytometric analysis

The expression of surface molecules on DC was analyzed by FACS (BDImmunocytometry Systems, San Jose, CA). For the staining of MHCclass-I (H-2Kb), MHC class-II (I-Ab), CD11c, CD40, CD80, and CD86,FITC- or PE-labeled rat mAbs were used. FITC- or PE-labeled rat IgG wassubstituted for specific Abs to obtain negative controls. All Abs were pur-chased from BD PharMingen.

MLR

Allogeneic T cells were separated from BALB/c mice by nylon wool col-umn procedures. The percentage of CD3-positive cells after the separation

was �85%, as assessed by FACS. After irradiation of DC, they were sus-pended in complete medium and seeded at 1 � 102 –2 � 104/well on96-well flat-bottom culture plates. The responder T cells were mixed withDC at 1 � 105/well and cultured for 5 days at 37°C, 5% CO2. During thelast 16–18 h of incubation, 1 �Ci/well of [3H]thymidine (New EnglandNuclear Life Science, Boston, MA) was added. Assays were performed intriplicate. On day 5, the cells were harvested and [3H]thymidine incorpo-rated into T cells was measured by a beta counter.

IL-12 ELISA

After day 7 DC had been cultured with or without tumor SN for 3 days, 1 �106 of DC were stimulated with a 200–400/1 dilution of SAC (v/v) for48 h. IL-12 p70 was assayed by an OptEIA ELISA kit (BD PharMingen).The threshold of the assay was 15 pg/ml.

Apoptosis assay

Cardiolipin located on inner mitochondria membranes was stained with10-nonylacridine orange (NAO; Molecular Probes, Eugene, OR) (13). Af-ter the samples had been adjusted to 1 � 106/500 �l, NAO was added at0.2 �g/ml and incubated for 15 min at 37°C. To evaluate mitochondrialmembrane potential, 1 � 106/500 �l of cells were stained with 10 �M ofpotential sensitive fluorescent dye, rhodamine 123 (Rh123; Calbiochem)for 15 min at 37°C (14). Cells were then washed and subjected to FACSanalysis.

The DNA content in samples was analyzed by staining with propidiumiodide (PI; Sigma) (14). After DC had been fixed with 50% ethanol for 30min at 4°C, the samples were treated with RNase A (Sigma) at 0.1 mg/mlfor 30 min at 37°C. Then, PI was added to samples at 0.1 mg/ml andincubated for 15 min at room temperature. The samples were subjected toFACS for cell cycle analysis. The population of hypoploidic DNA content(sub G0/G1 fraction) is determined to be apoptotic cells.

To protect DC from apoptosis, we treated day 7 DC with cytokines,caspase inhibitors, or some reagents before the addition of tumor SN. Theapoptosis assay was performed 24–72 h following treatment.

Caspase-3 assay

Caspase-3 activity in cells was measured by a FluorAce apopain assay kit(Bio-Rad, Hercules, CA). The assay is based on the detection of fluoro-genic substrates specifically cleaved by caspase-3. After 2 � 106 of day 7DC had been cultured in the presence or absence of SN, the lysates wereprepared by freeze-thaw. Caspase-3 activity was measured according to themanufacturer’s instructions.

Analysis of ceramide by mass spectrometry

Cellular lipids were extracted from at least 2 � 105 DC as reported pre-viously (13). Extracts were dissolved in chloroform/methanol (1:2, v/v)before analysis by mass spectrometry. Lipids were analyzed by direct in-fusion into a Quattro II triple quadrupole mass spectrometer (Micromass,Manchester, U.K.) as reported previously (13). Mass spectra were obtainedby scanning the range of 400–950 mass to charge ratio (m/z) every 1.6 sand summing individual spectra. The levels of ceramide were expressed asthe sum of the total ion currents (TIC) per cell for each of the ceramidespecies. In some samples, a similar analysis was performed under positivemode. For the analysis of sphingoid bases, the spectra were obtained byscanning 250–480 m/z every 0.7 s under positive mode. The comparisonof ceramide levels was performed among the series of samples on whichMS analysis had been performed on the same day under the same settings.

EMSA

Nuclear extracts or cytoplasmic protein were obtained from DC accordingto the methods described previously (15), with some modification. Thecells were suspended in lysis buffer containing 10 mM Tris-HCl (pH 7.5),1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF, 0.1 mMNa3VO4, and 0.1% Triton X-100. Then the samples were incubated on icefor 20 min and were centrifuged at 7500 � g for 10 min at 4°C. The pelletswere suspended in extraction buffer containing 20 mM Tris-HCl (pH 7.5),1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.1% Triton X-100, 25%glycerol, 0.5 mM EDTA, 0.5 mM PMSF, and 0.1 mM Na3VO4. After a30-min incubation on ice, they were centrifuged at 14,000 � g for 20 minat 4°C. The SN were obtained and used as nuclear extracts. The proteinconcentration was determined by a protein assay kit (Bio-Rad).

Two micrograms of extracts was incubated for 30 min in 20 �l of re-action buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 0.5 mMEDTA, 0.5 mM DTT, 50 mM NaCl, and 4% glycerol with [32P] end-labeled, double-stranded oligonucleotide probe specific for �B site (Pro-mega, Madison, WI) and 2.0 �g of poly(dI-dC) (Amersham Pharmacia

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Biotech, Piscataway, NJ). In some experiments, unlabeled NF-�B or mu-tant NF-�B oligonucleotide (Santa Cruz Biotechnology, Santa Cruz, CA)was added to the samples at 500-fold excess before the addition of labeledprobe. The complexes were resolved on 5% polyacrylamide gels in Tris-HCl (pH 8.0)-borate-EDTA buffer. Dried gels were placed with Kodak(Rochester, NY) OMAT x-ray film for 12–48 h at �70°C.

Western blotting

To isolate the whole cell lysates for immunoblotting, the cell pellets weresuspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mMNaCl, 0.6 mM PMSF, 0.1% SDS, and 1% Triton X-100. The samples wereincubated at 4°C for 30 min and then centrifuged at 14,000 � g for 30 min.The SN were collected, and total protein was quantified. Ten to 100 �g oflysate was separated by 10% SDS-PAGE and transferred to a polyvinyli-dene difluoride membrane blocked with 5% nonfat dry milk. Immunostain-ing was performed with rabbit polyclonal primary Abs specific for Bcl-xL/S (Santa Cruz Biotechnology) or Akt (Cell Signaling Technology,Beverly, CA) followed by incubation with goat polyclonal anti-rabbit IgGAb conjugated to HRP (Santa Cruz Biotechnology). Immunoreactive bandswere visualized using ECL reagents (New England Nuclear Life Science).

Akt kinase assay

Activity of Akt kinase was measured by an Akt kinase assay kit (CellSignaling Technology). Briefly, after DC had been cultured with or withoutSN for 3–4 days, whole cell lysates were collected and Akt was immuno-precipitated from �200 �g of lysate with immobilized mouse monoclonalanti-Akt Ab (1G1, IgG2a). Kinase reactions were performed at 30°C for 30min with precipitated Akt and 1 �g of GSK-3 fusion protein as substratesin the presence of 200 �M ATP. The proteins were separated by 10%SDS-PAGE and transferred onto polyvinylidene difluoride membranes.Phosphorylated GSK-3 was reacted with rabbit polyclonal anti-phospho-GSK-3�� (Ser21/9) Ab and HRP-conjugated anti-rabbit IgG Ab, and wasvisualized by LumiGro reagents (Cell Signaling Technology).

Statistical analysis

The Mann-Whitney U test was used to compare the values where appro-priate. Values of p � 0.05 were considered statistically significant.

ResultsTumor SN induces DC apoptosis

After the separation of cells on day 7, �90% of the cells exhibitedcharacteristic DC morphology. After culturing in the presence orabsence of tumor SN for an additional 3 days, no significant dif-ference in expression was seen for CD11c, CD40, CD80, CD86,H-2Kb, and I-Ab Ags among the groups (data not shown).

When tumor SN were added to day 7 DC and cultured thereafterwith GM-CSF and IL-4, the viability of B16SN-treated cells de-creased more rapidly than those in the other groups (data notshown). To test whether tumor SN induced DC apoptosis, westained cells with Rh123, NAO, or PI (13, 14). Among the celllines tested, B16SN exhibited the most potent proapoptotic activityon DC in these different assays after 72 h of incubation with SN(Fig. 1, A and B). Fibrosarcoma cell lines MCA207 and MCA102also showed a similar activity (Fig. 1B). The SN collected from themouse melanocyte cell line (Melan-A) and the fibroblast cell line(L cell) did not induce DC apoptosis (Fig. 1B), indicating thattumor SN-induced DC apoptosis is not related to the origin oftumor cells. For the remaining studies, we used the B16SN toexamine the mechanisms of tumor-induced DC apoptosis. Whenwe minimized the DC-B16SN incubation time, we determined thata 6-h exposure was sufficient to induce apoptosis after 48 h (datanot shown). This indicated that tumor-induced DC apoptosis wasexecuted by certain apoptotic signals, and not simply by the lackof nutrients.

DC exposed to B16SN display low MLR and low IL-12 p70production

To compare the ability of DC to stimulate T cell proliferation, anallogeneic MLR assay was performed. Day 10 DC treated with

B16SN exhibited a suppressed T cell response as compared withother SN (Fig. 2A). In addition, with the SAC stimulation, theproduction of IL-12 p70 from B16SN-treated day 10 DC was sig-nificantly lower than the other SN (Fig. 2B). These results sug-gested that the B16SN had an overall suppressive effect on T cellstimulatory activity of DC.

Ceramide accumulates in DC cultured with tumor SN

We analyzed lipid profiles of DC by negative and positive elec-trospray ionization mass spectrometry (ESI-MS). In the negativeion mode, the prominent peaks were found at m/z 572, 682, and684 (Fig. 3A). The other peaks at m/z 794, 796, and 885 wereidentified as phosphatidylcholine (PC)-chloride (Cl�) adducts (m/z794, 796) and phosphatidylinositol (PIn, m/z 885), respectively. InDC treated with B16SN, the relative amount of the 572, 682, and684 mass ions increased as compared with controls (Fig. 3),whereas PC and PIn did not significantly change regardless of thetreatments (Fig. 3). Similar results were obtained with DC treatedwith MCA207 or MCA102SN (data not shown).

Our earlier study identified the species at m/z 572 as C16 cer-amide-Cl� adduct (13). By analyzing the m/z 682 and m/z 684species using similar techniques and comparison to commercialstandards, they have been identified as C24:1 and C24:0 ceramide-Cl� adducts, respectively (data not shown). In the positive ionmode, the lipid species at m/z 725 was similarly determined to be

FIGURE 1. Tumor SN induces DC apoptosis. Day 7 DC were culturedwith or without 20% (v/v) Melan-A, L-cells, or tumor SN. A, DC apoptosisanalyzed by Rh123, NAO, or PI staining after 72 h of incubation with orwithout B16SN. M1 shows the region of apoptotic cells. A representativeresult from four series of experiments is shown. B, DC apoptosis assessedby NAO staining after 72 h of incubation with or without tumor SN. Re-sults are expressed as the mean � SD of three experiments. �, p � 0.05 vsSN�; ��, p � 0.01 vs SN�.

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sphingomyelin (SM)-sodium adduct (data not shown). The inten-sities of C16, C24:1, and C24:0 ceramides began to increase 24 hafter treatment with B16SN, reached their peaks by 48–72 h, andparalleled the increase in DC apoptosis (data not shown). The lev-els of SM tended to increase during this time period (Fig. 3C). Theaccumulation of C16 or C24 ceramides correlated well with thedegree of DC apoptosis induced by various tumor SN (correlationcoefficient of TIC and percentage of apoptosis, 0.906). These re-sults indicated that C16 and/or C24 ceramide accumulation wasclosely related with tumor-induced DC apoptosis. To exclude thepossibility that the increase of ceramide is a result of apoptosis, weexamined lipid profiles from day 9 DC that were deprived of GM-CSF and IL-4 for 2 days. Under these conditions, apoptosis oc-curred in �40% of the DC. However, neither C16 nor C24 cer-amide increased as compared with controls (data not shown),suggesting that ceramide is not generated as a result of apoptosis.

Ceramide mediates tumor-induced DC apoptosis

To examine whether ceramide induces DC apoptosis, we added C2ceramide or its analog C2 dihydroceramide to day 7 DC and ex-amined apoptosis after 24 h. C2 ceramide, but not C2 dihydroce-ramide, induced apoptosis in DC (Fig. 4A).

To investigate whether the elevation of endogenous ceramidecould induce DC apoptosis, we treated DC with the acid cerami-dase inhibitor, NOE (16), or the glucosylceramide synthase inhib-itor, PDMP (17). Under ESI-MS analysis, both of these reagentsincreased C16 and C24 ceramide levels in DC (Fig. 4B) in a dose-dependent fashion (data not shown). Such treatments led to DCapoptosis (Fig. 4C), thus demonstrating that the increase of en-dogenous ceramide induces DC apoptosis. When these inhibitorswere used in the presence of B16SN, additive effects were ob-served in enhancing ceramide levels and DC apoptosis with en-hanced kinetics as early as 24 h after treatment (Fig. 4, B and C).Such additive effects were also observed with MCA207 SN (datanot shown). Thus, some tumor SN increase endogenous ceramidelevels in DC, which then contribute directly to the induction ofapoptosis.

Ceramide can be generated by either SM breakdown induced byacid or neutral sphingomyelinase (SMase) and/or by de novo cer-

amide synthesis (10). To block acid or neutral SMase, we usedchlorpromazine (18) or glutathione (19), but failed to prevent tu-mor-induced DC apoptosis (data not shown). However, when DCwere treated with L-cycloserine, an inhibitor of serine palmitoyl-transferase (20), ceramide levels and the degree of apoptosis werereduced (Fig. 5, A and B). The ability of L-cycloserine to preventtumor-induced DC apoptosis was abrogated by the addition of C2ceramide (Fig. 5B). These results indicate that the increase of cer-amide that is involved in tumor-induced apoptosis is generated viathe de novo synthesis pathway. FB1, an inhibitor of ceramide syn-thase (21), also reduced ceramide mass (Fig. 5A); however, it didnot prevent DC apoptosis (Fig. 5B). It has been reported that theinhibition of ceramide synthase increases sphingoid bases (sphin-ganine) that are growth inhibitory and cytotoxic to some cells (22).In B16SN-treated DC, the sphinganine level was higher than thatin control DC (TIC per cell of C16 sphinganine, 0.45 vs 0.26). FB1(50 �M) increased sphinganine (TIC, 10.0) and apoptosis (45%) inDC in the presence of B16SN (Fig. 5B). However, the addition of100 �M L-cycloserine to FB1-treated culture reduced both sphin-ganine and apoptosis to baseline levels (TIC, 0.29; apoptosis 22%)in B16SN-treated DC. These results demonstrate that the accumu-lation of sphinganine is critically involved in the failure of FB1 toprevent DC apoptosis, despite the reduction of C16 and C24ceramides.

Tumor-induced DC apoptosis is caspase independent

To test whether a caspase-dependent pathway is involved in DCapoptosis, caspase-3 activity was measured. Caspase-3 activitywas higher in B16SN-treated DC than in untreated cells (Fig. 6A).On day 7, DC were pretreated with 25–50 �M caspase-3 inhibitorAc-DEVD-CHO or caspase-1 inhibitor Z-VAD-fmk before the ad-dition of tumor SN. Although the increase of caspase-3 activity inB16SN-treated DC was suppressed by these inhibitors (data notshown), neither of these rescued DC from B16 or MCA207SN-induced apoptosis (Fig. 6B). It is well known that agonisticanti-Fas Ab (clone CH-11) induces apoptosis in Jurkat cells in acaspase-dependent fashion. To confirm that the concentration ofthese caspase inhibitors is sufficient to block caspase-dependent

FIGURE 2. DC exposed to B16SN display low allogeneic MLR and low IL-12 p70 production. Day 7 DC were incubated with or without 20% (v/v)tumor SN for 3 days. A, Allogeneic MLR. �, p � 0.05 vs SN�. B, DC were stimulated with a 200/1 dilution of SAC for 48 h. IL-12 p70 in culture SNwas measured by ELISA. �, p � 0.05 vs SN�, SAC�.

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FIGURE 3. Increase of ceramides in tumor-induced DC apoptosis. Day 7 DC were incubated with or without 20% (v/v) B16SN for 3 days. Lipid samplesextracted from the same numbers of DC were analyzed by the negative ESI-MS. The profiles of DC without SN (A) and those with B16SN (B) are alignedin the same scale of y-axis. x-axis, m/z; y-axis, percent relative abundance. C16, C24:1, and C24:0 ceramides are as chloride adducts. PC�Cl, PC-chlorideadduct. C, TIC of ceramides in DC are expressed as the mean � SD of three experiments. �, p � 0.05 vs SN�.

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apoptosis, we treated Jurkat cells with 200 ng/ml anti-Fas Ab (CH-11; Upstate Biotechnology, Lake Placid, NY) in the presence orabsence of these inhibitors. After 24 h of incubation, 75% of Jurkatcells were apoptotic by NAO staining in the absence of the inhib-itors. Both Z-VAD-fmk (25 �M) and Ac-DEVD-CHO (25 �M)strongly inhibited anti-Fas-induced Jurkat cell apoptosis (apoptoticcells, 7 and 28%, respectively). These results indicate that the in-hibitors were sufficient to prevent caspase-dependent apoptosis.Therefore, tumor SN-induced DC apoptosis is mainly executed bya caspase-independent mechanism(s).

Tumor-induced DC apoptosis is reversed by TPA

Several molecules are known to improve DC survival, such as LPS(23, 24), TNF-� (25), IL-12 (7), TNF-related activation-induced cy-tokine (6), and CD40 ligand (25). We used 10–100 ng/ml LPS, 10–50ng/ml IL-12, or 10–50 ng/ml recombinant TNF-� to rescue DC fromtumor-induced apoptosis. We stimulated DC by cross-linking CD40with hamster anti-mouse CD40 mAb and subsequent anti-hamsterIgG Ab (26). However, these reagents failed to improve DC survival

in the presence of tumor SN (data not shown). These reagents had nosignificant effect on NOE- or PDMP-induced DC apoptosis, suggest-ing that ceramide-mediated DC apoptosis is resistant to these survivalfactors (data not shown).

Phorbol ester, TPA, is a potent activator of serine/threonine ki-nases, such as PKC, ERK, or PI3K, and a known antagonizer ofsome types of ceramide-mediated cell death (27). We added 10ng/ml TPA to DC 15 min before the addition of B16 or MCA207SN. Our results indicated that TPA protected DC from SN-inducedapoptosis (Fig. 7A) and delayed the decrease of viable DC for upto 6 days (Fig. 7B). Furthermore, TPA decreased DC apoptosiscaused by NOE, but did not in PDMP-treated DC (Fig. 7A). TPAsuppressed ceramide increases induced by tumor SN or NOE, butnot by PDMP (Fig. 7C). These results demonstrate that TPA isefficacious for the blocking of tumor-induced or ceramide-medi-ated DC apoptosis. Such protection by TPA correlated with thereduction of ceramide levels, suggesting that the inhibition of cer-amide accumulation is one of the mechanisms of preventing DCfrom tumor-induced apoptosis.

FIGURE 4. Exogenous and endogenous ceramides induce DC apoptosis. A, Day 7 DC incubated with or without C2 ceramide (C2) or C2 dihydroce-ramide (Dihydro-C2) for 24 h. B and C, Day 7 DC cultured in the presence of 20% (v/v) B16SN and 50 �M each NOE or PDMP for 24 h. The same volumeof vehicle (DMSO) was added to control DC. A, DC apoptosis examined by NAO staining. Results shown are the mean � SD from three experiments.�, p � 0.05 vs SN�. B, TIC of C16 and C24:1 ceramides in DC. Representative data from three experiments are shown. C, DC apoptosis examined byNAO staining. Results shown are the mean � SD from three experiments. �, p � 0.05 vs SN�.

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Next, we examined which pathway was involved in DC survivalby using various kinase inhibitors. Treatment with the PI3Kinhibitor, LY294002, led to DC apoptosis in a dose-dependentfashion (Fig. 8A). Alternatively, treatment with the ERK inhibitor(PD98059) or the p38 SAPK inhibitor (SB23085) also led to DCapoptosis, but to a much lower level than LY294002 (Fig. 8A). ThePKC inhibitor, bisindoylmaleiamide-I had no effect on DC apo-ptosis (Fig. 8A). When 10 ng/ml TPA was used before these in-hibitor treatments, DC apoptosis induced by LY294002 or

PD98059 was reduced, but that induced by SB23085 was en-hanced (Fig. 8A). These data indicate that the PI3K pathway iscritically involved in DC survival, and ERK and p38 SAPK path-ways are at least partially involved. In addition, TPA is able toactivate PI3K and ERK but not p38 SAPK in DC. To examinewhich pathway was responsible for TPA-induced protection againsttumor-induced DC apoptosis, we pretreated DC with kinase inhibitorsbefore the addition of TPA and tumor SN. The protective effect ofTPA was abolished with LY294002, suggesting that activation of

FIGURE 6. Tumor-induced DC apoptosis is caspase independent. A, After day 7 DC were incubated with or without 20% (v/v) tumor SN, cell lysateswere collected from DC and their caspase-3 activity was measured. �, p � 0.05 vs SN�. B, Day 7 DC were pretreated with 25 �M each caspase-3(Ac-DEVD-CHO) or caspase-1 (ZVAD-fmk) inhibitor before the addition of 20% (v/v) B16SN. Apoptosis was examined by NAO staining after 48 h.Results are expressed as the mean � SD of three experiments. �, p � 0.05 vs SN�.

FIGURE 5. L-cycloserine prevents DC from tumor-induced apoptosis by inhibiting increases in ceramide levels. Day 7 DC were treated with FB1 orL-cycloserine (L-cy) and were cultured with or without 20% (v/v) B16SN for 3 days. In some samples, C2 ceramide (C2) or C2 dihydroceramide(Dihydro-C2) was added to the culture. The same volume of vehicle (ethanol) was added to control DC. A, TIC of C16 and C24:1 ceramides in DC.Representative data from three experiments are shown. B, DC apoptosis examined by PI staining. Results shown are the mean � SD from three experiments.�, p � 0.01 vs SN�; ��, p � 0.05 vs B16SN.

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PI3K is one of the mechanisms TPA uses in preventing DC apoptosis(Fig. 8B). Conversely, the inhibition of PI3K is a major facet of tu-mor-induced DC apoptosis.

Tumor SN or endogenous ceramide suppresses Akt, NF-�B, andbcl-xL in DC

One of the downstream targets of PI3K is Akt/protein kinase B(28). We examined Akt kinase activity in DC treated with or with-out tumor SN. Akt activity decreased in DC incubated withB16SN, whereas the expression of Akt did not differ (Fig. 9). Adownstream Akt target is NF-�B, an essential transcription factorthat up-regulates multiple survival genes in many types of cells(29). EMSA revealed that NF-�B activation was suppressed inB16SN-treated DC (Fig. 10A). The binding of the probe to thesamples was inhibited with consensus but not with mutant NF-�Boligonucleotide, indicating that the binding is specific for the �Bsequence (Fig. 10A). In Western blot analysis, the expression of

nuclear NF-�B p50 and Rel-B in B16SN-treated DC was lowerthan those in untreated DC (data not shown), also confirming thesuppression of NF-�B activity. The Bcl-2 family of genes playspivotal roles in protecting a variety of cells from ceramide-medi-ated apoptosis (30). The expression of bcl-xL in B16SN-treatedDC was reduced compared with controls (Fig. 10B), whereas bcl-2expression did not differ between the two conditions (data notshown).

To simulate tumor-induced ceramide increase, we treated DCwith NOE and PDMP. The Akt activity, the NF-�B activation, orthe expression of bcl-xL in DC was suppressed by NOE or PDMP(Figs. 9 and 10), suggesting that intracellular ceramide down-reg-ulates Akt, NF-�B, and bcl-xL. The treatment of DC withLY294002 also impaired these molecules (Figs. 9 and 10), con-firming that Akt, NF-�B, and bcl-xL are downstream of PI3K. Thepretreatment of DC with TPA or L-cycloserine before the additionof B16SN restored NF-�B and bcl-xL levels (Fig. 10), which

FIGURE 7. TPA prevents tumor-induced DC apoptosis by inhibiting increases in ceramide. Day 7 DC were pretreated with 10 ng/ml TPA for 15 minand were cultured subsequently for 2 days with or without 20% (v/v) tumor SN or 50 �M NOE or PDMP. The same volume of vehicle (ethanol) was addedto control DC. A, DC apoptosis assessed by NAO staining. Results were expressed as the mean � SD of three experiments. �, p � 0.05 vs SN�; ��, p �0.05 vs DC treated with relevant SN or reagents. B, Viable cells were analyzed from day 7 by the MTT assay. The percentages of viable DC are shownby the ratio of OD at 570 nm to that on day 0. �, p � 0.05 vs B16SN. C, TIC of C16 and C24:1 ceramides in DC. Representative results from three seriesof experiments are shown.

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indicated that these molecules are up-regulated by the reduction ofceramide and/or the activation of PI3K. Therefore, these resultsdemonstrate that ceramide accumulates in DC exposed to sometumor SN, which subsequently down-regulates PI3K and its down-stream Akt kinase, bcl-xL, and NF-�B. All of these changes reducethe survival potential of DC and subsequently enhance the suscep-tibility of DC to apoptosis.

DiscussionExtensive research has been focused on the pleiotropic roles ofceramide in a variety of biological phenomena, such as prolifera-tion, differentiation, cellular senescence, and apoptosis (10). In thisstudy, we showed that DC become apoptotic in the presence ofcertain tumor SN. Simultaneously, DC were prevented from pro-liferating or became arrested in cell cycle progression, as shown bydecreased uptake of [3H]thymidine (data not shown). DC that wereprimed to be apoptotic were unable to support T cell responses,despite their matured phenotypes. From these observations, we

hypothesized that the accumulation of ceramide mediates tumor-induced DC apoptosis.

We demonstrated the involvement of ceramide in DC apoptosisthrough several lines of evidence. First, ceramide accumulated inDC incubated with tumor SN, which resulted in DC apoptosis andcorrelated well with the degree of apoptosis. Second, the accumu-lation of endogenous ceramide by treatment with inhibitors of cer-amide metabolism induced DC apoptosis. Third, these inhibitorsenhanced ceramide mass and accelerated apoptosis in the presenceof tumor SN. Finally, the blocking of ceramide synthesis withL-cycloserine prevented DC from tumor-induced apoptosis.

We used ESI-MS to analyze each of the ceramide species in DC(13). ESI-MS of lipids offers several advantages over existingtechniques: 1) total lipid profiles are able to be analyzed withoutany prior purification or chemical derivatization, 2) collision-in-duced dissociation of the lipids allows direct confirmation of theirstructure, and 3) changes in structure are able to be assessed di-rectly at the molecular level (13). Using this approach, we previ-ously reported that C16 ceramide is up-regulated during the laterphases of apoptosis induced by ionizing radiation or Fas ligation inmultiple cell types (13). In this study, we identified C16, C24:1,and C24:0 ceramides as the predominant species that increased intumor-induced apoptotic DC as determined by the TIC of each ofthe ceramide species. Like previous studies including our own (31,32), we used synthetic ceramide as internal standards and con-firmed the proportional relationship between the TIC of each of theceramide species and their ratio to standards (data not shown). Incontrast with C16 or C24 ceramide, PC levels did not change re-gardless of the treatments. Thus, the comparison of ceramide lev-els by TIC as performed in this study reflects an accurate assess-ment of ceramide levels.

It has been reported that ceramide changes not only quantita-tively but also qualitatively in some stress responses (33). In ap-optotic DC, increases in C24:1 and C24:0 ceramides were moredistinctive than C16 ceramide, which was opposite to that seen inJurkat T cells (13). Such differences may be explained in part by

FIGURE 9. Tumor SN or endogenous ceramide down-regulates Akt ki-nase. Day 7 DC were cultured for 3 days with or without 20% (v/v)B16SN, 25 �M LY294002 or 50 �M NOE. Two hundred micrograms ofcell lysates was subjected to Akt kinase assay and 50 �g of lysates toWestern blot, respectively. GSK-3 protein phosphorylated by Akt wasprobed by specific anti-phospho-GSK-3�� Ab and anti-rabbit IgG Ab(top). Akt was detected with anti-Akt Ab and anti-rabbit IgG Ab (bottom).Abbreviations are shown in Fig. 10.

FIGURE 8. TPA attenuates tumor-induced DC apoptosis by activating PI3K pathway. A, Day 7 DC were treated with various kinase inhibitors with orwithout pretreatment of 10 ng/ml TPA. DC apoptosis was assayed by NAO staining after 2 days. The same volume of vehicle (DMSO) was added to controlDC. LY, LY294002; PD, PD98059; SB, SB23085; Bis, bisindoylmaleiamide I. �, p � 0.05 vs SN�; ��, p � 0.01 vs SN�; ���, p � 0.05 vs 25 �M LY.B, Day 7 DC were treated with various kinase inhibitors for 2 h. Subsequently, DC were given 10 ng/ml TPA 15 min before the addition of 20% (v/v)B16SN and cultured for 2 days. DC apoptosis was examined by NAO staining. �, p � 0.05 vs SN�; ��, p � 0.05 vs B16SN.

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the different patterns of lipid profiles based on cell type (31). How-ever, it should be noted that each of the ceramide species may havedifferent biological activities. The proapoptotic activity of C16 andC24 ceramide was evidenced in prior studies in addition to ourown (13, 34). On the contrary, some metabolites of ceramide maysignal proliferative and anti-apoptotic responses in some cells.Sphingosine-1-phosphate, which is generated from ceramide bythe action of ceramidase and sphingosine kinase, has proliferativeactivity and antagonizes ceramide-induced apoptosis (35). Thus,mass spectrometric analyses of lipid profiles are necessary to iden-tify each species involved at the molecular level.

Many researchers use cell-permeable short chain (C2 or C6)ceramides to mimic the influences of ceramide on cells in vitro(10). We confirmed that C2 ceramide induced DC apoptosis in adose-dependent manner. However, it is still unclear whether ex-ogenous ceramide analogs accurately mimic the effects of intra-cellular ceramide (36). Some studies showed that exogenous cer-amide acts through different sites of action from endogenousceramide in terms of caspase activation and apoptosis (36, 37).Thus, the data obtained with such ceramide analogs need to beevaluated carefully. A balance between synthesis and metabolismof ceramide through multiple enzymes determines intracellularceramide levels. As a substitute for short chain ceramide, we usedinhibitors of acid ceramidase (NOE) or glucosylceramide synthase(PDMP) to elevate endogenous ceramide levels in DC. In sometypes of cells, these inhibitors cause ceramide accumulation by theinhibition of ceramide metabolism and then induce apoptosis (16,17). With concomitant incubation with tumor SN, these inhibitorsaccelerated apoptosis and ceramide accumulation in DC as com-pared with those with SN alone. Therefore, the pathways involvingthese enzymes are activated in DC to metabolize accumulated cer-amide in the presence of tumor SN. In contrast, tumor-derivedfactors may have an inhibitory activity on these pathways.

The direct downstream targets of ceramide may be different ac-cording to cell type, magnitude of ceramide generation, and/or itslocalization. Ceramide is synthesized mainly by the SMase path-way and the de novo synthesis pathway (10). The enzymes in-

volved in these pathways reside in different intracellular compart-ments, causing different localizations of ceramide (10, 36). Thus,it is conceivable that cells may respond differently to some formsof stress by generating ceramide from different sources (38, 39).From the sequential analysis of SM mass and the inability ofSMase inhibitors to prevent DC death, the involvement of thispathway in DC apoptosis may be unlikely.

Ceramide is also synthesized from sphingosine or sphinganinethrough the action of serine palmitoyltransferase and ceramidesynthase (10, 36). Recently, additional studies implicated that cer-amide generated via the de novo pathway has a signaling functionin apoptosis (40, 41). Perry et al. (40) reported that this ceramidepathway plays a regulatory role in mediating membrane-relatedapoptotic events, which were independent of the caspase activationcascade. Most of the signals transduced by the de novo pathwayare unknown; however, Raf-1/ERK has been recently reported asits direct downstream target (41). In this study, L-cycloserine waseffective in preventing tumor-induced DC apoptosis, but FB1 wasnot. Several explanations may be possible for such differences inactivity. First, FB1 is a mycotoxin and possesses proapoptotic ac-tivity in some cell types (42). It has been reported that blockingceramide synthase with FB1 results in the increase of sphinganine,which is cytotoxic to some cells (22). In our study, sphinganinelevels, as well as apoptosis, increased in DC with the FB1 treat-ment in the presence of B16SN. The combination of L-cycloserineand FB1 reduced sphinganine levels and apoptosis, demonstratingthe involvement of sphinganine in FB1-induced enhancement ofB16SN-induced DC apoptosis. FB1 exhibited a toxic effect on DCat concentrations as low as 50 �M, whereas L-cycloserine wastolerated up to 200 �M (data not shown). Second, serine palmi-toyltransferase is the initial and rate-limiting enzyme in the path-way and governs the production of ceramide (10, 40). L-cy-closerine prevents C16, C24, and sphinganine increases in DC,thus resulting in the protection of DC from tumor-induced apo-ptosis. Based on these data, the putative tumor-derived factor isconsidered to stimulate the de novo pathway in DC, thus leadingto increases in endogenous ceramide levels.

FIGURE 10. Tumor SN or endoge-nous ceramide down-regulates NF-�B andbcl-xL in DC and TPA or L-cycloserine re-verses them. Day 7 DC were cultured for 3days with or without 20% (v/v) B16SN, 25�M LY294002, 50 �M (A) or 100 �M (B)NOE, or 50 �M PDMP. In some samples,DC were pretreated with 10 ng/ml TPA or100 �M L-cycloserine before the additionof B16SN. A, Two micrograms of nuclearextracts from DC were given a labeledNF-�B probe and separated on 5% poly-acrylamide gel. Unlabeled consensus(oligo) or mutant oligonucleotide (mutantoligo) was added to the samples at 500-foldexcess and incubated for 30 min before theaddition of the probe (left panel). L-cy,L-cycloserine; Hela, extracts from Helacells. Š, p50-p50 homodimers; 4, p50-p65 or p50-RelB, p50-cRel heterodimers.B, Ten micrograms of cell lysates collectedfrom DC was separated on 10% polyacryl-amide gel. Bcl-xL expression was exam-ined by Western blot.

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Our study shows that tumor-induced DC apoptosis occurs via acaspase-independent pathway, or at least is not critical for DCapoptosis. One of the mechanisms of caspase-independent apopto-sis is the disruption of mitochondria (43). Ceramide has directeffects on isolated mitochondria and its functions, resulting inmembrane permeability transition and generation of reactive oxy-gen species, which are followed by loss of membrane potential(10). In the present study, the involvement of reactive oxygen spe-cies as an effector in DC apoptosis is unlikely, because the scav-enging agent or mitochondrial respiratory chain inhibitor (44) didnot prevent tumor-induced DC apoptosis (data not shown).

Tumor-induced or ceramide-mediated DC apoptosis was notfully reversed by other DC survival factors, such as LPS, IL-12,TNF-�, or agonistic CD40 Ab. These results suggested that tumor-induced DC apoptosis is executed by unique and multifactorialmechanisms. The inability of TNF-� or CD40 Ab to rescue DCfrom tumor- or ceramide-induced apoptosis may be related to theirpotential to increase endogenous ceramide levels in DC (12). Wefound that phorbol ester, TPA, is the most effective in protectingDC from tumor-induced apoptosis. The protective effect of TPA inour system was mostly attributed to its ability to activate PI3K toreduce intracellular ceramide mass. The importance of PI3K orERK pathways for survival or protection from apoptosis has beendemonstrated in many types of cell, including human or murineDC (23, 24). Downstream of PI3K, Akt executes anti-apoptoticeffects by way of several mechanisms, such as phosphorylation ofBc1-2/Bc1-xL-associated death promoter, activation of NF-�B,up-regulation of bcl-2 or bcl-xL, or maintaining the integrity ofmitochondria (28). Whereas some cytokines or growth factors areknown to activate PI3K, stress-induced or exogenous ceramide hasbeen reported to suppress the PI3K/Akt pathway (45, 46). Also inthis study, we confirmed that an endogenous ceramide increase isable to suppress Akt activity by treating DC with NOE. In DCpretreated with TPA or L-cycloserine, apoptosis was attenuatedand the expression of NF-�B and bcl-xL were restored, suggestingthe importance of these molecules for DC survival in the presenceof tumor SN. Thus, it is conceivable that the ceramide increaseinduces DC apoptosis by the inhibition of the PI3K/Akt and/orERK-dependent survival pathways, not by the enhancement ofcaspase-dependent apoptotic pathways. In our hands, bcl-2 expres-sion did not differ in the presence of tumor SN. Different roles orsites of action have been implicated between bcl-2 and bcl-xL inceramide-mediated apoptosis (47).

We found that several human tumor cell lines induced matura-tion and subsequent apoptosis in human monocyte-derived DCwithin 24–72 h of incubation (data not shown). Kiertscher et al.(48) reported similar findings when tumor SN was added to DCfrom the beginning of culture. In our study, ceramide accumulatedbefore the occurrence of apoptosis in human DC incubated withtumor SN (data not shown). These results imply the possibility ofceramide playing an essential role in tumor-induced DC apoptosisin both murine and human systems.

The question remains as to the nature of the tumor-derived pro-apoptotic factor. It is well known that TNF-� or Fas ligand (FasL)is an inducer of apoptosis in various types of cells, accompanied bythe elevation of ceramide. The possibility that TNF-� is respon-sible for the observed effects is low, because the addition ofrTNF-� did not lead to DC apoptosis and anti-TNF-� Ab did notrestore tumor-induced apoptosis (data not shown). Murine BM-derived DC are reported to express both Fas and FasL. BM DCgenerated from Fas knockout (lpr) mice were also susceptible totumor SN-induced apoptosis, suggesting that Fas-FasL interactionis not required (data not shown). Biochemical analyses withB16SN are currently underway to identify tumor-derived factors.

In summary, our study indicates that some tumor cells produceproapoptotic factors that act on BM DC. Tumor-induced DC ap-optosis is mediated by ceramide, which down-regulates PI3K, ac-companied by the suppression of NF-�B and bcl-xL. TPA protectsDC from apoptosis by activating PI3K and/or enhancing ceramidemetabolism. It has been demonstrated that the longevity of DC invivo is necessary to prime and maintain effective anti-tumor im-mune responses, which may be attained by protecting DC fromapoptosis. Transduction of anti-apoptotic genes into DC is oneapproach to meet these needs (8). Recently, metabolizing endog-enous ceramide by the transduction of acid ceramidase genes ordirect administration of sphingosine-1-phosphate has been shownto attenuate TNF-� or radiation-induced apoptosis (16, 49). There-fore, the modulation of ceramide synthesis or ceramide-metabo-lizing pathways needs to be considered as potential therapeuticmanipulations to prevent DC apoptosis in the clinical setting.

AcknowledgmentsWe thank Richard E. Redlinger, Jr. for his assistance in preparing themanuscript. We also thank Dr. Elieser Gorelik (University of Pittsburgh)for fruitful discussions and providing the Melan-A cell line.

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