tumor necrosis factor-asensitizes prostate...

[CANCER RESEARCH 59, 1606–1614, April 1, 1999]

Tumor Necrosis Factor-a Sensitizes Prostate Cancer Cells tog-Irradiation-induced Apoptosis1

Kotohiko Kimura, Cai Bowen, Sarah Spiegel, and Edward P. Gelmann2

Division of Hematology/Oncology, Department of Medicine [K. K., C. B., E. P. G.], and Department of Biochemistry and Molecular Biology [S. S.], Lombardi Cancer Center,Georgetown University, Washington, DC 20007-2007

ABSTRACT

LNCaP prostate cancer cells are highly resistant to induction of pro-grammed cell death byg-irradiation and somewhat sensitive to the death-inducing effects of tumor necrosis factor (TNF)-a. Simultaneous exposureof LNCaP cells to TNF-a and 8 Gy of irradiation was synergistic andresulted in a 3-fold increase of apoptotic cells within 72 h compared toTNF-a alone. It appeared that TNF-a sensitized the cells to irradiationbecause, when cells were irradiated 24 h after exposure to TNF-a, in-creased cell death was observed. In contrast, irradiation delivered 24 hprior to TNF- a exposure did not result in more cell death than afterTNF-a alone. TNF-a induced expression of its own mRNA, but TNF-amRNA induction was neither induced nor enhanced by irradiation. Acti-vation of the transcription factor nuclear factor kB can be induced byTNF-a and has a modulating antiapoptotic effect. But enhancement ofTNF-a-induced cell death by irradiation did not result from alteredactivation of nuclear factor kB. TNF-a treatment of LNCaP cells resultedin partial activation of caspase-8 and -6 but not caspase-3. There was onlyminimal poly(ADP-ribose) polymerase cleavage seen in LNCaP cells afterexposure to both TNF-a and irradiation at 72 h, a time when 60% of thecells were apoptotic. Experiments with peptide inhibitors of cysteine andserine proteases suggested that caspases were the predominant mediatorsof apoptosis induced by TNF-a alone but that serine proteases contributedsignificantly to cell death induced by TNF-a plus irradiation. TNF- aincreased production of ceramide in LNCaP cells 48 h after exposure.Although irradiation alone had no effect on ceramide production inLNCaP cells, TNF-a plus irradiation induced significantly more ceramidethan TNF-a alone. Ceramide production did not occur immediately afterexposure to TNF-a, but rather was delayed such that ceramide levels wereincreased only 24 h after exposure to apoptotic stimuli. Moreover, non-toxic levels of exogenous C2-ceramide sensitized LNCaP cells to irradia-tion similarly to TNF- a, suggesting that one mechanism by which LNCaPcells were sensitized to irradiation was by increased intracellular ceram-ide. Hence, ceramide generation is a critical component in radiation-induced apoptosis in human prostate cancer cells. Inhibition of ceramidegeneration may provide a selective advantage in the development ofradioresistance in prostate cancer.

INTRODUCTION

One mechanism by which cancer cells become resistant to radiationor chemotherapy is disruption of the pathways that lead to apoptosis(1, 2). Radiation is an important treatment modality for prostatecancer, and it kills cells by inducing apoptosis, generating free oxygenspecies, and causing DNA strand breaks (3–6).g-Irradiation activatesacidic sphingomyelinase to produce ceramide, a catabolic product ofmembrane sphingolipids that is a cell death signal (7–11). Ceramidecan also be generatedde novoby ceramide synthase, as was shown inLNCaP cells induced to undergo apoptosis after exposure to 12-O-

tetradecanoylphorbol-13-acetate (12). Although ceramide appears tofunction as a second messenger for stress signaling (13–17), there isstill some controversy about the precise role that ceramide plays inapoptosis (7, 18–21).

LNCaP is a hormone-dependent human prostate cancer cell linethat is moderately sensitive to induction of apoptosis by TNF-a,3 aninflammatory cytokine that can induce a diverse range of biologicalresponses (22, 23). Signaling by TNF-a is initiated by binding totumor necrosis factor receptor 1, which causes the association of anadapter protein TNF receptor-associated death domain with the intra-cellular death domain of the tumor necrosis factor receptor 1 molecule(24). TNF receptor-associated death domain mediates the subsequentrecruitment of an adapter protein Fas-associated death domain to forma DISC, which initiates apoptosis through activation of caspase-8, aproximal element in the cascade of cysteine proteases (18, 25–30).TNF-a can induce ceramide production, which, in turn, inducesactivation of caspase-3 and -7, which cleave PARP, and of caspase-6,which cleaves lamin B, thus contributing to dissolution of the nuclearenvelope (19, 31–33). Signaling by TNF-a also activates antiapop-totic cell signals via activation of the transcription factor NF-kB,which may override the effects of apoptosis pathways in some cells(34–36).

Here, we show that, although LNCaP cells are highly resistant tog-irradiation-induced apoptosis, irradiation and TNF-a synergize toinduce apoptosis in LNCaP cells. Cell death after exposure to TNF-aplus irradiation is characterized by increased production of ceramide48–72 h after exposure. Moreover, induction of cell death by thecombination ofg-radiation and TNF-a is mediated by the cooperativeeffects of cysteine and serine proteases, both of which have to beinhibited to block LNCaP cell death completely.

MATERIALS AND METHODS

Cell Culture. Human prostate cancer cell line LNCaP was routinely cul-tured at 37°C in IMEM (Biofluids, Rockville, MD) supplemented with 5%FCS using standard cell culture procedures (37, 38). Experiments in serum-freemedium were performed in IMEM supplemented with either insulin, selenium,and transferrin at 5mg/ml each or with 1 mg/ml BSA. Twenty-four h beforeexposure to TNF-a and/or irradiation, the medium was changed to IMEMwithout phenol red (Biofluids) supplemented with 5% charcoal-stripped calfserum. TNF-a was used at 40 ng/ml to treat LNCaP cells except whendose-response studies were performed. Caspase inhibitors were added 1 hbefore treatment of cells with either TNF-a or g-irradiation. ExogenousC2-ceramide was added to cells cultured in serum-free IMEM for 24 h. YVADwas purchased from Bachem Bioscience (King of Prussia, PA). DEVD andzVAD were purchased from Enzyme Systems Products (Livermore, CA).TLCK was purchased from Sigma Chemical Co. (St. Louis, MO). Recombi-nant, human TNF-a, produced inE. coli a and purified by standard chromato-graphic techniques was purchased from Boehringer Mannheim (Indianapolis,

Received 7/15/98; accepted 1/28/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by NIH Grant CA57178 and an award from the CaPCUREFoundation (to E. P. G.), NIH Grant CA61774 (to S. S.), and American Cancer SocietyGrant BE275 (to S. S.).

2 To whom requests for reprints should be addressed, at Division of Hematology/Oncology, Department of Medicine, Lombardi Cancer Center, Georgetown University,3800 Reservoir Rd, NW, Washington, DC 20007-2007. Phone: (202) 687-2207; Fax:(202) 784-1229; E-mail: [email protected].

3 The abbreviations used are: TNF, tumor necrosis factor; DISC, death-inducingsignaling complex; PARP, poly(ADP-ribose) polymerase; NF-kB, nuclear factorkB;IMEM, improved MEM; YVAD, N-acetyl-Tyr-Val-Ala-Asp-chloromethylketone; DEVD,N-acetyl-Asp-Glu-Val-Asp(OMe)-CH2F; zVAD, z-Val-Ala-Asp(OMe)-CH2F; TLCK,Na-p-tosyl-L-lysine-chloromethylketone; ISEL,in situ end labeling; PMSF, phenylmeth-ylsulfonyl fluoride; RT-PCR, reverse transcriptase-PCR;GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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IN). The primary structure of recombinant, human TNF-a is identical to thatof natural human TNF-a and has aMr of 17,000. According to the manufac-turer, the specific activity is 108 units/mg. C2-Ceramide was purchased fromBiomol Laboratories (Plymouth Meeting, PA). Forg-irradiation, we used a JLShepherd Mark I Irradiator137Cs source with a dose rate of 209 cGy/min.

Apoptosis Assays.ISEL assay is routinely used in our laboratory forapoptosis determination and has been described previously(39). To detectDNA ladder formation, we suspended cells in 50 mM Tris-HCl (pH 8.0), 10mM EDTA, and 0.5% sodium-N-lauroylsarcosinate. RNase and proteinase Kwere added to the cell suspension both at the concentration of 10mg/mlsequentially. After incubation with RNase for 1 h and with proteinase K for4 h, the samples were extracted several times with phenol-chloroform andprecipitated with ethanol and salt. The precipitants were dissolved in TE buffer[10 mM Tris-HCl (pH 8.0)-1 mM EDTA] and resolved on a 1.8% agarose gel.

Western Blotting. Two hundredmg of total cellular protein were re-solved by electrophoresis on either 8 –16% or 10 –20% SDS-polyacryl-amide gradient gels and transferred onto nitrocellulose membranes(Trans-Blot transfer medium; Bio-Rad Laboratories, Hercules, CA). Afterblocking with 5% milk in 10 mM Tris-HCl (pH 8.0)-150 mM NaCl with0.05% Tween 20, membranes were probed with monoclonal antibody toPARP (Enzyme Systems Products, Dublin, CA), rabbit antiserum tocaspase-3 (a gift from Kristine Kikly, SmithKline Beecham, King ofPrussia, PA), caspase-8 antiserum (from the laboratory of Peter Krammer),or antibody to lamin B (Calbiochem, La Jolla, CA) and visualized withenhanced chemiluminescence detection (Pierce, Rockford, IL).

Ceramide Assay.Lipids were extracted according to the method of Blighand Dyer (40). Cell lysates were suspended into methanol:chloroform:H2O(1:1:1). The organic phase was dried under N2. Lipids were resuspended in 40ml 7.5% octyl-b-D-glucofuranoside (Calbiochem, Cambridge, MA), 5 mM

cardiolipin (Avanti Polar-Lipids, Alabaster, AL), and 10 mM imidazole (pH6.6) by freezing and thawing several times. Lipids were radiolabeled by thekinase reaction usingsn-1,2-diacylglycerol kinase (Calbiochem) and[g-32P]ATP (Amersham Life Science, Arlington Heights, IL). One hundredmlof 100 mM imidazole-HCl (pH 6.6), 100 mM NaCl, 25 mM MgCl2, and 2 mM

EGTA; 20 ml of 20 mM DTT; 30 ml of 6.6 mM ATP; 10 mCi of [g-32P]ATP;and 10mg of sn-1,2-diacylglycerol kinase were added to each suspended lipidsample. After the reaction mixture was incubated at room temperature for 1 h,the reaction was extracted with 0.5 ml of chloroform:methanol:HCl (100:100:1) and 50ml of 2 M KCl. Lipids were separated by TLC, and theradioactive bands were quantitated using a Molecular Dynamics PhosphorIm-ager analyzer (Sunnyvale, CA). The amount of ceramide content in eachsample was standardized based upon the amount of phospholipids determinedby the method of Van Veldhoven and Mannaerts (41).

NF-kB Mobility Shift Assay. Binding of NF-kB to a consensus DNAsequence was performed as described by Dignamet al. (42). Cells werewashed with ice-cold PBS, resuspended in 5 volumes of 1 mM Hepes-HCl (pH7.5), 1.5 mM MgCl2, 10 mM KCl, 0.25 mM DTT, and 1 mM PMSF andincubated on ice for 20 min. Cells were homogenized by 15–20 strokes in aglass homogenizer and centrifuged at 7500 rpm for 6 min at 4°C. The pelletswere washed and resuspended in 20 mM Hepes-HCl (pH 7.5), 20% glycerol,0.42M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.25 mM DTT, and 1 mM PMSF,vortexed, and centrifuged at 14,000 rpm for 45 min. Twomg of nuclear extractwere assayed for binding to an oligonucleotide containing a radiolabeledNF-kB binding site consensus sequence (Santa Cruz Biotechnology, SantaCruz, CA). Binding was carried out by mixing 1–2ml of nuclear extract; 2mlof 50 mM Hepes-HCl (pH 7.5), 5 mM EDTA, 5 mM 2-mercaptoethanol, and20% glycerol; 1ml of 1.5 mg/ml poly(dIzdC); 1ml of 20 mM Hepes-HCl (pH7.9), 20% glycerol, 100 mM KCl, 250 nM EDTA, 500 nM DTT, 500 nM PMSF,and 1% NP40; 1ml of oligonucleotide; and 3ml of water. The samples wereincubated for 30 min at a room temperature. The samples were resolved on 4%acrylamide gels and analyzed by PhosphorImager.

RT-PCR. TNF-a, TNF-b, and controlGAPDH mRNA were assayed byPCR amplification of cDNA isolated from LNCaP cells. The primer sequencesfor PCR amplification are as follows:TNF-a, left, 59-GGCTCCAGGCGGT-GCTTGTTCC-39, and right, 59-CAGGCTTGTCACTCGGGGTTCG-39;TNF-b, left, 59-GGTCCAGCTCTTCTCCTCCCAGTA-39, and right, 59-GC-GAAGGCTCCAAAGAAGACAGTA-39; GAPDH, left, 59-GGGAAGGT-GAAGGTCGGAGT-39, and right 59-GGGGTCATTGATGGCAACAA-39.PCRs were cycled at 94°C for 1 min, 55°C for 1 min, and 72°C for 30 s. For

TNF-a, 32 cycles were completed; forTNF-b, 37 cycles were completed; andfor GAPDH, 32 cycles were completed.

RESULTS

Induction of Apoptosis in LNCaP cells by TNF-a and g-Irra-diation. LNCaP cells are resistant to apoptosis after exposure to 8 Gyof irradiation. TNF-a at a concentration of 40 ng/ml induced;30%apoptotic cells by 72 h (22, 37). However, simultaneous exposure ofLNCaP cells to TNF-a and 8 Gy of irradiation resulted in.60%apoptosis after 72 h (Fig. 1A). A similar enhancement of cell death bycombined TNF-a and irradiation treatment was seen in serum-freemedium (Fig. 1B). To determine whether irradiation affected thesensitivity of LNCaP cells to apoptosis induced by TNF-a, we ex-posed the cells to different concentrations of TNF-a and, simulta-neously, to 8 Gy of irradiation. Concentrations of TNF-a of at least 10ng/ml were required for the induction of apoptosis, whether or not thecells were irradiated with 8 Gy, and the range of effective TNF-aconcentrations for apoptosis induction was not affected byg-irradia-tion (Fig. 1C). No apoptosis was seen at lower doses of TNF-a. Toconfirm the induction of apoptosis, we also examined DNA fragmen-tation. Substantially more DNA ladder formation was seen aftertreatment with TNF-a and irradiation than after TNF-a alone (Fig.1D). The degree of DNA ladder formation generally correspondedwith the results of the ISEL assay shown in Fig. 1C. We also observedthat a higher dose ofg-irradiation (20 Gy) neither caused apoptosis inLNCaP cells nor enhanced the effect of TNF-a any more than did 8Gy of irradiation (Fig. 1E). The temporal relationship of exposurewith TNF-a and irradiation was also shown to be important for theenhancement of TNF-a-induced apoptosis. If LNCaP cells were ex-posed to TNF-a 24 h after 8 Gy of irradiation, no enhancement ofapoptosis was seen compared to exposure to TNF-a alone. In contrast,if the cells were first exposed to TNF-a and irradiated 24 h later, thesynergistic effect of the two treatments was seen (Fig. 1F). Thecombined effect of TNF-a and irradiation was seen when cells wereirradiated at 12 or 24 h but not at 36 h after treatment with TNF-a(data not shown).

Expression of Endogenous TNF-a. Because TNF-a can induceits own mRNA, we asked whether irradiation affected the induction ofTNF-a message and, thereby, enhanced cell death activated by exog-enous TNF-a. Fig. 2 shows a semiquantitative RT-PCR assay forTNF-a mRNA under different conditions used to treat LNCaP cells.TNF-b (lymphotoxin) andGAPDHwere used as controls for mRNAinduction and loading. Whereas TNF-a induced its own mRNA,irradiation did not induceTNF-a mRNA. Moreover, combinedTNF-a and irradiation did not change the expression ofTNF-amRNA that was induced by TNF-a alone.

Activation of NFkB in LNCaP Cells Exposed to TNF-a andg-Irradiation. NF-kB is an antiapoptotic transcription factor that isreleased from a cytoplasmic complex with its inhibitor protein, IkB,after binding of TNF-a to its receptor (34–36, 43). After TNF-atreatment, there were no differences in nuclear NF-kB in the presenceor absence of 8 Gy of irradiation (Fig. 3). NF-kB was found to bepresent in the nucleus at 6 h after treatment with TNF-a and sustainedat the same level throughout 48 h of exposure to TNF-a with orwithout irradiation (data not shown).

Caspase Activation in LNCaP Cells Exposed to TNF-a andg-Irradiation. Proteolytic cleavage and activation of caspase-8 isbelieved to result from interaction between caspase-8 and the DISCafter TNF-a binds to the TNF receptor (27). Caspase-8 cleavagepeptides p42 and p20 were generated by 72-h treatment with 40 ng/mlTNF-a, with or without irradiation (Fig. 4). There was a slightinduction of caspase-8 cleavage induced by 10 ng/ml TNF-a after 8

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TNF-a AND RADIATION-INDUCED APOPTOSIS



Gy of irradiation. Caspase-3 is downstream of caspase-8 and is acritical effector protease for cleavage of PARP and for activation ofthe endonuclease that generates characteristic DNA ladder formationseen in some forms of cell death (44, 45). We observed essentially nocaspase-3 activation in LNCaP cells treated with either TNF-a aloneor TNF-a and 8 Gy of irradiation. In contrast, exposure to 30 nM

okadaic acid, a known apoptotic agent for these cells, resulted inrobust caspase-3 activation (Refs. 46 and 47; Fig. 4).

A small amount of PARP cleavage was seen after cells wereexposed to TNF-a alone at 40 ng/ml, a concentration that induced30% apoptosis at 72 h (Fig. 4). PARP cleavage activity was

increased in cells treated with 40 ng/ml TNF-a and 8 Gy ofirradiation for 72 h. For comparison, treatment of LNCaP cellswith 30 nM okadaic acid for 48 h resulted in pronounced PARPcleavage. Because relatively little PARP cleavage was seen, it wasnot surprising that another protease that cleaves PARP, caspase-7,was not activated, as is the case after exposure of LNCaP cells tolovastatin (Refs. 48 –52; Fig. 4).

Cleavage of lamin B by caspase-6 is important for dissolution of thenuclear membrane during apoptosis (53, 54). Cleavage of lamin B intothe characteristicMr 28,000 fragment was detected under the sameconditions that generated caspase-8 activation (Fig. 4). Western blot-

Fig. 1. Effect of TNF-a and g-irradiation onapoptosis in LNCaP cells.A, time course of theapoptosis in LNCaP cells treated with 40 ng/mlTNF-a and 8 Gy of irradiation.B, effect of 40ng/ml TNF-a and 8 Gy on apoptosis in LNCaPcells after 72 h in serum-free medium.C, doseresponse of apoptosis to TNF-a 72 h after exposureto either 0 or 8 Gy of irradiation.D, DNA ladderformation in LNCaP cells treated with differentamounts of TNF-a and 8 Gy ofg-irradiation.LaneC, positive control; contains DNA from LNCaPcells treated with 30 nM okadaic acid for 48 h. A50-bp DNA ladder marker is in theleft-most lane.E, dose response tog-irradiation of apoptosis withor without treatment of 40 ng/ml TNF-a after 72 h.F, timing of TNF-a exposure and irradiation deter-mine the degree of apoptosis. LNCaP cells weretreated with 40 ng/ml TNF-a, 8 Gy of irradiation,or both at either the beginning of the experiment orat 24 h. Apoptosis was assayed 72 h after thebeginning of the experiment.Bars, SD.

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ting of extracts from cells treated with lower doses of TNF-a or 8 Gyirradiation alone showed only intact lamin B, indicative of a lack ofactivation of caspase-6.

Inhibition of Apoptosis by Protease Inhibitors. Apoptosis inprostate cancer cells may be mediated by both caspases and serineproteases (55). To determine the role of both cysteine and serineproteases in apoptosis induced by TNF-a and 8 Gy of irradiation, we

treated cells with peptide inhibitors specific for either caspases orserine proteases (18, 56). DEVD, an inhibitor of caspase-3, -7, and -8(21), at 50mM completely inhibited apoptosis induced by TNF-aalone but had almost no effect on apoptosis induced by TNF-a plus 8Gy of irradiation (Fig. 5A). YVAD, an inhibitor of caspase-1 andrelated proteases (19), did not show any effect on apoptosis of LNCaPcells after treatment with TNF-a in the absence or presence ofirradiation (Fig. 5B). zVAD, which is a general inhibitor of caspaseactivity (21), completely inhibited TNF-g-induced apoptosis in LN-CaP cells and reduced apoptosis induced by TNF-a plus 8 Gy ofirradiation by 75% (Fig. 5C). Because caspase inhibition by zVADdid not completely abrogate apoptosis induced by TNF-a plus 8 Gyirradiation, we also treated LNCaP cells with an inhibitor of serineproteases, TLCK. TLCK had no effect on apoptosis induced byTNF-a alone but substantially reduced apoptosis after exposure toTNF-a and 8 Gy irradiation (Fig. 5D). The importance of bothcaspases and serine proteases to apoptosis induced by TNF-a plus 8Gy irradiation was shown by the combined inhibitory effect of bothpeptides that was greater than either peptide used alone (Fig. 5E).

To confirm that the peptide inhibitors inhibited caspase activity,caspase-8 and PARP cleavage was determined in the presence of thedifferent inhibitors. Caspase-8 activation was completely blocked byzVAD and DEVD (Fig. 6). The fact that caspase-8 activation wasblocked although substantial apoptosis occurred in the presence ofcaspase inhibitors underscored the fact that that serine and other

Fig. 2. RT-PCR analysis ofTNF-a, TNF-b, and GAPDH mRNA in LNCaP cellstreated with 40 ng/ml TNF-a with or without irradiation. As a control, 100 nM etoposidewas used, which induces partial cell death in LNCaP cells (39) and complete cell death inTSU-Pr1 prostate cancer cells. As can be seen, induction ofTNF-a mRNA occurred bothin LNCaP cells and in TSU-Pr1 cells 48 h after treatment with etoposide.

Fig. 3. A mobility shift assay for NF-kB in LNCaP cells treated with TNF-a and/org-irradiation. LNCaP cells were treated with 40 ng/ml TNF-a and/or 8 Gy of irradiation,as indicated. The nuclear extracts were made 48 h after the treatment, and a mobility shiftassay for NF-kB was performed. The competitor inLane 7 (from left to right) was100-fold excess of cold oligonucleotide with the NF-kB binding sequence. The p65 andp50 protein components of NF-kB are indicated on theright.

Fig. 4. Effect of TNF-a and/org-irradiation on the cleavage of caspase-8, caspase-3,caspase-7, lamin B, and PARP. LNCaP cells were treated with different concentrations ofTNF-a without or with 8 Gy of irradiation. Protein extracts were made after 72 h andsubjected to Western blotting.Lane C, positive control protein samples. For caspase-8, thepositive control was Jurkat cells treated with anti-FAS antibodies. For the other Westernblots, theLane Ccontained proteins from LNCaP cells treated with 30 nM okadaic acidfor 48 h.

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proteases mediated apoptosis induced by the combination of TNF-aand irradiation.

Low levels of PARP cleavage seen in LNCaP cells treated withTNF-a and irradiation were abrogated by treatment with DEVD,zVAD, and also TLCK, suggesting that the low level of PARPcleavage seen after exposure to TNF-a plus irradiation resulted fromcaspase activation downstream from serine proteases (Fig. 6). Inter-estingly, lamin B cleavage, which is attributed to caspase-6 (53), wasnot inhibited by DEVD or TLCK but was blocked by zVAD. Because

DEVD inhibited caspase-8 activation but not lamin B cleavage, thissuggests that caspase-6 is not directly activated by caspase-8, as hasbeen suggested previously (57). The experiments with caspase inhib-itors showed that, althoughg-irradiation alone had no effect onprotease activation or apoptosis, when combined with TNF-a, g-irra-diation augmented caspase activation and caused serine protease ac-tivation. Thus, both caspases and serine proteases are responsible forthe execution of cell death in LNCaP cells exposed to TNF-a andirradiation.

Fig. 5. Effect of caspase inhibitors on LNCaP cells treated with 40 ng/ml TNF-a, irradiation, or both.A–D, cells were treated with the indicated concentrations of inhibitors in theabsence or presence of 40 ng/ml TNF-a and 8 Gy irradiation.E, after treatment with either zVAD, TLCK, or zVAD plus TLCK, cells were then exposed to 40 ng/ml TNF-a and 8Gy of irradiation. The percentages of apoptotic cells in A-E were determined after 72 h by the ISEL assay.Bars, SD.

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Ceramide Formation after Exposure to TNF-a and/or g-Irra-diation. Both TNF-a andg-irradiation can induce the production ofceramide in a wide variety of cells (7, 58). Exogenous ceramide itselfcan induce cells to undergo apoptosis (59, 60). In some cells, ceramideformation was induced as early as 5–10 min after exposure to TNF-a.However, there were no significant changes of ceramide levels duringthe first 60 min after exposure of LNCaP cells to TNF-a and/or 8 Gyof irradiation (data not shown). Although there was no increase inceramide production after 6 h, there was a 2-fold increase afterexposure of LNCaP cells to 40 ng/ml TNF-a for 24 and 48 h (Fig.7A). Whereas 8 Gy of irradiation alone did not induce any ceramideproduction in LNCaP cells, in the presence of TNF-a, there was amarked increase in ceramide levels. However, because no increase inceramide production was seen within 6 h ofexposure to irradiation orTNF-a, it is unlikely that activation of ceramide production is an earlyevent in apoptosis induced by either TNF-a alone or in the presenceof 8 Gy of irradiation. In LNCaP cells, YVAD, DEVD, TLCK, andzVAD had very little effect on ceramide production after exposure toTNF-a or to the combination of TNF-a and irradiation (Fig. 7B). Theminimal effect of protease inhibitors on ceramide production is incontrast to the marked inhibition of apoptosis (Fig. 5E) and caspaseactivation (Fig. 6), suggesting that ceramide production is upstream ofserine protease and caspase activation.

Because ceramide production induced by TNF-a may possiblysensitize LNCaP cells to induction of apoptosis byg-irradiation, weexposed LNCaP cells to increasing doses of the cell permeable C2-ceramide and measured apoptosis. At a concentration of 10mM,C2-ceramide induced 15% apoptosis after 72 h, which was markedlyincreased to 48% after simultaneous exposure tog-irradiation (Fig. 8).Therefore, both TNF-a and C2-ceramide sensitized LNCaP cells toirradiation and synergized with irradiation in the induction of celldeath.

DISCUSSION

Cancer cells develop resistance to programmed cell death as amechanism of chemotherapy and radiation resistance. LNCaP prostatecancer cells are virtually resistant to induction of programmed celldeath by exposures tog-irradiation as high as 60 Gy.4 Although DNA

damage at high doses of radiation precludes clonogenicity, resistanceto apoptosis may permit prostate cancer cells to survive lower dosesof g-irradiation to allow DNA repair and further replication. It isnoteworthy that the LNCaP cells used in this study have no mutationsof the p53 gene and, therefore, must not be susceptible to p53-mediated apoptosis after irradiation (61). The observation that irradi-ation did not result in caspase activation or ceramide productionsuggests that LNCaP cells have a very early block in radiation-induced apoptosis. This block may be attributed to attenuated ceram-ide production, as our data have suggested, but may also involve otherdeath pathways. Our preliminary investigations have suggested thatmitochondrial death pathways are not activated by TNF-a plus irra-diation in LNCaP cells.4

TNF-a enhancedg-irradiation-induced apoptosis in LNCaP cells atleast in part through the augmentation of pathways involving ceram-ide. Ceramide production increased 24 h after exposure to TNF-a withor without irradiation. Apoptosis was seen at 48 h. Unlike many othercell lines that undergo cell death within a few hours after exposure to

4 K. Kimura and E. P. Gelmann, unpublished data.

Fig. 6. Effect of caspase inhibitors on the cleavage of caspase-8, PARP, and lamin B.After LNCaP cells had been treated with DEVD, TLCK, or zVAD, the cells were furthertreated with 8 Gy of irradiation in the absence or presence of 40 ng/ml TNF-a. After 72 h,cell lysates were subjected to Western blotting. In the lamin B blot, the cleavedMr 28,000band is designated by thearrow. For caspase-8, the positive control (Lane C) was Jurkatcells treated with anti-FAS antibodies. For the other Western blots,Lane Ccontainedproteins from LNCaP cells treated with 30 nM okadaic acid for 48 h.

Fig. 7. Production of ceramide after exposure of LNCaP cells to TNF-a with or withoutirradiation. A, ceramide content in LNCaP cells was measured 6, 24, and 48 h aftertreatment of the cells with 40 ng/ml TNF-a and/or 8 Gy of irradiation.Bars, SD.p, astatistically significant difference between the result for TNF-a plus radiation at 48 hcompared to TNF-a alone. Significance determined by Student’st test at a statistical errorof 0.5% (73).B, after LNCaP cells had been treated with YVAD, DEVD, TLCK, orzVAD, the cells were further treated with 40 ng/ml TNF-a and/or 8 Gy of irradiation.Ceramide content was measured 48 h after the treatment.

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FAS ligand, such as Jurkat, LNCaP cells take 48–72 h to show arobust cell death response to TNF-a (62). Etoposide and okadaic acidalso require 48–72 h to initiate cell death in LNCaP cells (63, 64),whereas staurosporine can induce apoptosis within 12 h of exposure(63).

We observed that caspase-8 was activated after exposure of LNCaPcells to TNF-a, but only minimal activation of caspase-3 and PARPcleavage, both of which are downstream from caspase-8, could bedetected. In contrast, caspase-7 was not activated, even after TNF-aand irradiation. Similarly, other prostatic cancer cells are resistant tochemotherapy and TNF-a because they lack caspase-3-like activity(65). The very low level of PARP cleavage in LNCaP cells afterexposure to TNF-a and irradiation is further evidence that neithercaspase-3 nor caspase-7 was substantially activated. This is in agree-ment with the speculation that a relatively low level of endogenouscaspase-3 activity may be responsible, in part, for the limited degreeof apoptosis induced by TNF-a in LNCaP cells (65).

Caspase-8 cleavage was induced by TNF-aa and was markedlyenhanced after exposure to 8 Gy of irradiation. DEVD blocked bothcaspase-8 cleavage and apoptosis induced by TNF-a. DEVD alsoblocked caspase-8 cleavage after exposure to TNF-a plus irradiationbut had virtually no effect on apoptosis. TLCK, a serine proteaseinhibitor, partially blocked both caspase-8 cleavage and apoptosis

after exposure to TNF-a plus irradiation. TNF-a directly activatescaspase-8 by signal transduction through the DISC. However, whenTNF-a was combined with irradiation, caspase-8 was activated boththrough the DISC and by serine protease activation. Moreover, al-though DEVD blocked caspase-8 cleavage, it had minimal effect onapoptosis after exposure to the combination of TNF-a and irradiation.

Our data show that increased ceramide production correlated withenhanced apoptosis in LNCaP cells treated with TNF-a and irradia-tion. Although irradiation alone did not alter ceramide levels, in thepresence of TNF-a, it increased ceramide production. In many cells,ceramide is induced rapidly by both TNF-a andg-irradiation. (7, 18).In LNCaP cells, we could not detect any change in ceramide contentwithin the first 12 h after TNF-a treatment or irradiation. By 24 h afterexposure to TNF-a with or without irradiation, an increase in ceram-ide content was observed. The ceramide increase at 24 h preceded theonset of substantial apoptosis in LNCaP cells. In cells that manifestboth immediate and late peaks of ceramide production, apoptosiscorrelates with the magnitude of the later rise of ceramide (19). Ourdata agree with these findings in that the timing and magnitude ofceramide production correlated with the degree of cell death. More-over, it seems that ceramide generation is a critical component ofradiation-induced apoptosis in human prostate cancer cells and sug-gests that blockage of ceramide generation may provide a selectiveadvantage in the development of radioresistance of prostate tumors.These data are in accordance with recent results indicating that loss ofceramide production confers resistance tog-irradiation-induced apop-tosis in WEHI-231 cells (66).

There appears to be a limit to the amount of apoptosis that can beinduced by TNF-a in LNCaP cells. We observed that exogenousTNF-a activated its own mRNA but that irradiation had no effect onTNF-a production. This suggests that limitations in the death responseafter exposure to TNF-a are probably mediated at the level of inter-action between TNF-a and its receptor. In contrast to the failure ofLNCaP cells to activate TNF-a mRNA in response to irradiation,radiation-induced death of sarcoma cell lines was mediated, at least inpart, by TNF-a acting through an autocrine pathway (67). There isalso evidence that cell death induced by radiation or chemotherapymay be accompanied by autocrine production of death ligands (68,69).

Other death ligands may be activated in response to irradiation.Recent data demonstrated that ceramide rapidly and strongly inducedexpression of Fas ligand, which interacts with CD95 (APO-1/Fas) toactivate cleavage of caspases in fibroblasts (70, 71). Activation ofCD95 (APO-1/Fas) signaling by ceramide mediated cancer therapy-induced apoptosis (71). Thus, ceramide, similarly, may linkg-irradi-ation to induction of the CD95 system of apoptosis to enhance andamplify the death program in an autocrine manner. LNCaP cellsexpress CD95 antigen, but when exposed to FAS antibody in mediumcontaining 10% FCS, they have a minimal apoptotic and antiprolif-erative response to anti-Fas antibody (72). Our experiments wereperformed in 5% charcoal-stripped calf serum, conditions that mayhave been more permissive for the effects of Fas ligand to contributeto cell death after TNF-a and irradiation. However, we saw nodifference in death induction when our experiments were done inserum-free medium.

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1999;59:1606-1614. Cancer Res Kotohiko Kimura, Cai Bowen, Sarah Spiegel, et al. -Irradiation-induced Apoptosis

γ Sensitizes Prostate Cancer Cells to αTumor Necrosis Factor-

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