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~skBAissfijojrtjcjzxps/186k15 , Intl. Catmer: 000, 1-9 (1998) O 1998 Wile y-Liss, Inc. nnli~o-, ea me M.msmna tn+o„ Ap6roi Cup., Puafuaun tl. nMYw, InlrMbn.M Can1n 1. C.rc.r REGULATION OF GI/S TRANSITION AND INDUCTION OF APOPTOSIS IN HL-60 LEUKEMIA CELLS BY FENRETINIDE (4HPR) Amra M. DtPO;TawrrroNto, Tze-chen Hsmt, Susan C. OLSON and Joseph M. Wu' 2505442176 Departntent ojBiochentisfry and Molecular Biology, New York Medical College. Yalhallq NY, USA We previously reported that all-trons rednoic acid (RA) and fenretlnide (4HPR) suppress HL-60 leukemia cell growth and cau:e partial cell arrest In the G,-to-5 phase. Moreover, 4HPR but not RA induces apoptosis In HL-60 cells. To Investigate further the observed biological effects, cyclin DI and cdk4 expression and the level of phosphorylatlon of the retlnoblas- toma protein Rb were assessed. Cyclin D I and cdk4 expres- sion and Rb phosphorylatlon were significantly reduced, by 40-75X, after 24 hr of treatment with RA or 4HPR; these decreases were either transient, e.g., only at 24 hr for cdk4, or sustained for 72 hr. In general, more pronounced decreases were seen in the 4HPR-treated cells. Evidence for 4HPR- induced apoptosis comes from (1) cleavage of the enzyme poly(ADP-rlbose) polymerase (PARP) to an 89-kDa trun- cated product, (2) appearance of DNA ladders on agarose gel electrophoresis, and (3) higher Incorporation in siW of digoxi- genin nucleoddes Into the free 3'-ends of DNA. Overntght pretreatment with 0.5-5.0 pM of the CPP32Inhibltor DEVD, but not the ICE Inhlbitor YVAD, significantly reduced the specific proeessing of PARP, suggesting that CPP32 Is In- volved In the mechanism of action of 4HPR. Analysis of 2 Ilpid-derFed second messengers, ceramlde and diacylglycerol (DAG), s a function of time of treatment with RA or 4HPR, showed ceramlde but not DAG to be significantly albeit transiently Increased 2-fold at 3 hr, by 4HPR. To test further whether ceramide may be Involved In the signaling cascade that culminates In the induction of apoptmis In 4HPR-treated HL-60 cells, the effects of fumenisin Bi, an inhibitor of ceramide synthase, were studied. Simultaneous treatment of cells with 4HPR and 25-100 pM furrtonistn Bi resulted In a dose-dependent reduction in the elevation in ceramide, the extent of PARP cleavage, and inductlon of apoptasis Pretreat- ment with DEVD or WAD, on the other hand, had no effect on the 4HPR-induced Increase In ceramide. Inc f. Cancer 77t00-00, 1998. O 7998 Wilry-Cirr, Iac. ' Previously, we reported that RA and 4HPR suppress growth of HLr60 leukemia cells, an effect that was accompanied by partial cellular arrest in the G1-t.o-S phase of the cell cycle. We also showed by flow cytomettic analysis that 4HPR, ttnlike RA, induces apoptosis in the treated cells (DiPietrammnio et at, 1996). To gain additional insights into the observed biologic effects of 4HPR, we examined the expression of several proteins participating in G,/S contml, such as cyclin Dl, cdk4, and pRb. Our results show Utat treatment with either RA or 4HPR resulted in a decrease in pRb, and correspondingly cyclin D I and cdk4, which may at least in patt account for the observed Gi arrest effects. A transient increase in the processing of (poly(ADP-ribose)polymerase (PARP) to the apoptosis-characteristic truncated products was also observed in 4HPR-iteated cells. By employing inhibitors that preferentially inhibit either CPP32 (DEVD) or intedeukin-15 converting enzyme (ICE) (YVAD) (Na et aL, 1996; Hasegawa er al., 1996), the cleavage of PARP may be ascribed to CPP32. Additional data in support of the induction of apoptosis by 4HPR included (1) the appearance of DNA ladders on agarose gels with DNA isolated from 4HPR and not from control or RA-treated cells, and (2) immunobistochemical analysis wherein the relative abundance of free 3'-ends in the DNA was approximated by labeling with the terminal deoxynucleotidyl transferase. 4HPR treatment led to an initial rise in the level of ceramide, which was effectively abolished by the simultaneous addition of fumonisin B„ an inhibitor of ceramide synthasc (Badiani et aL, 1996; Witty et at. 1996), whereas treatment with DEVD or YVAD had no effect. Cells treated with 4HPR and 25-100 pM fumonisin B, also showed a dose-dependent reduction in PARP cleavage, and the extent of apoptosis. These results suggest that 4HPR acts by triggering an elevation in ceramide as an early event, followed by activation of CPP32, and its subsequent biochemical sequelae to culminate in the induction of apoptosis. Natural and chemically synthesized derivatives of vitamin A (tetinoq display diverse pharmacological and biological activities, including regulation of cell proliferation, differentiation and pro- grammed cell death, also known as apoptosis (Love and Gudas, 1994; Greenberg and Sporn, 1996; Hong and Sporn, 1997). In animal models, retinoids have been reported to prevent formation of epithelial and mesenchymal tumors (Moon et at, 1994). Moreover, trarts-retinolc acid (RA), has demonstrated efficacy for ttea6ment of acute pmmyelocytic leukemia in humans (Warrell rt al., 1991; Degos, 1992). Bmad use of retinoids, however, has been restricted by its hepatotoxic and teratogenic effects when used at high concentrations (Spom and Roberts, 1984; Lippman et at, 1987). N-(-4-hydroxyphenyUtetinamide (4HPR), a relatively new synthetic analog of RA, has emerged as a retinoid with greater clinical potentials by virtue of the fact that it exhibits increased antieateinogenic activity yet reduced toxicity. 4HPR has been shown to induce apoptosis in several malignant cell types (Di Vinci et aC, 1994; Delia et aL, 1995; Wang and Phang, 1996; Robertson et at, 1997; Chan et aL, 1997), and has regyted chemopreventive properties in tissue culture and animal studies (Naik ct al., 1995; Moon and Constan[inou, 1997; Cooaway ct aL, 1998). Accotd- ingly, 4HPR is currently being evaluated in several human clinical trials for treating a variety of cancets including that of the breast and the prostate (Greenwald et aL, 1993; Costa et aL, 1994; ICelloff et at, 1994; Kienta et at, 1997). The mechanism of action of 4HPR, however, has not been elucidated. MATFRIAI-AND MEfHODS Chemicals RA was from Sigma (St. Louis, MO) and 41)PR was supplied by the Johnson Pharmaceutical Research Institute (Spring House, PA). The 2 chemicals were dissolved in absolute ethanol, as a 10 and 1 mM stock, respectively. The ceramide synthase inhibitor fumonisin B, obtained from Calbiochem (San Diego, CA), end the cell- petmeable ICE inhibitors YVAD-CHO and DEVD-CHO, pur- chased from Biomol (Plymouth Meeting, PA), were dissolved in distilled water or DMSO as a 1.4 mM and 0.5 M stocks, respectively. Cel! cufmre Human promyelocytie HLr60 and the breast cancer MCF-7 cells from the ATTC (Rockville, MD), were cultute.d in RPMI 1640 media containing L-glutamine supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml). Typically, 2 X 105/ml HL60 cells and 1 X IW/ml MCF-7 cells were seeded in T-75 tlasks as 10 ml cultures. On the indicated days, cells were Grent spoosort: the Vivian Wu-Au Memorial Cancer Res<arch Fund; the Philip Morris Co. J 'Concapoodeoce to: Deps[nneot of Biechemisoy and Molecular Biol- ogy New Yark Medical College, Sssic Sdemr Buildiog, Vtlhslla, NY 10595, USA- Fex: (914).594-4(158. E-mdl:Ioseph_Wu®nymc.rdu

/^Mb: LC (CeaceC) - YAUb: 1 SbSJ: 9 UU l YU7: 1 Do JuD 1S 1.3:J9:42S 1NY8 L,t '' A1HssAjqYtjcjxxps/186k15 2 © 250 200 G v~ O 150 a °o y u a~ w .~ o \ 100 U 50 24 DIPIEflUMOMO EfAL f ® .~.~.~.~.~ :~.~.~.~. 48 . Time (hours) CODtrol 3µMRA 3 µM 4HPR 72 Ptatnt6 1-Changes in cycGu DI and cdk4 expression in response to neatment witb RA or 4FIPR Expression of cyclin Dl and cdkl was determined by Westetn blot .nalysis, using extracts ftom control and treated ce1Ls. (a): Quantification of cyclin DI cxprrssion. (b): Qu.ntlfication of chd4 protein expression. The expression of various ptoteins was based on the desitomettic tracing of the Western blot data, with eonttol valons set at 100 Densitomettic analysis utilired a software ptognm purchased from Jandel Scieutific (Coxte Msdere, CA). Results are the mean of 2 exneriment<.

1illihfE : IJO (Gancer) - PAGE: 3 SESS: 9OUI'PUT: Tfirc-Jau'23 23:59:48 1998 Lt .$/Iissrjcjfijcjxxps7186k15 i PIF3OTROPIC FFFEC[S OF 4HPR IN HUMAN LEUKEMIA CFLL4 24 h 48 h 72h 1 2 3 4 5 6 7 8 9 pRb-) Rb--10' FIGURE 2-Deerease in pRb expression in retinoid neated cells. The methods and analysis of Western blots Rb and pRb protein expression in unireated (lanes 3, 6, and 9) HL6(/ cells were identical to that described in the legend of Figure I. harvested, washed twice with PBS and counted using a hemocymm- eter. Cell viability was determined using Trypan blue exclusion. Treatment with fmnonisin Bt and ICE inhibitors Typically 5 X 105 cells/ ml were seeded and pretreated overnight with fumonisin Bi (FBI), or with the cell-permeable ICE inhibitors YVAD-CHO and DEVD-CHO, prior to the addition of 413PR Cell growth was monitored for up to 24 hr and the presence of PB, YVAD-CHO or DEVDLHO in the culture media did not alter cell proliferation. Assessment of apoptosis in HG60 cells Agamse gel electrophoresis of DNA. DNA was isolated from control and 4HPR-treated cells, using the Qiagen (Chatsworth, CA) QlAamp method, modified as follows. Following lysis of the cells using the buffer provided by the manufacturer, the lysate was incubated with RNase A/protease K solution, and then mixed with 0.2 N NaOH, 1% SDS, followed by the addition of 7.5 M ammonium acetate to precipitate protein, large DNA and RNA After centrifugation to remove the precipitated ngcleic acid, the supernatant was transferred to a new tube and mixed with 0:6 vol isopropanol to precipitate low m.w. DNA. The precipitated DNA was washed with 70% ethanol, dried, dissolved in 10 mM Tris-HCI, 1 mM EDTA, pH 8.0 and electrophoresed in 1.8% agarose gels. The gel was stained for visualization of DNA banding patterns. Detection of apoptosis by labeling free 3,;-endt with digoxigenin nucleotides and tenninal deoxynucleotide transferase. Apoptosis was also measured using the ApopTag in situ apoptosis detection kit according to the manufacturer's instructions (Oneor, Gaithers- burg, MD). Control and 4HPR-treated cells were mounted on slides and apoptosis was assessed microscopically. Protein extraction Cells were suspended in lysis buffer (50 pl buffer/106 cells) containing 62.5 mM Tris-HCI (pH 6.8), 6.0 M utea, 2% SDS, 10% glycerol. 5% S-mercaptoethanol, 0.00125% bromophenol blue, I mM DTT, 0.5% NP-00, 0S mM PMSF, and 10 pg/ml of each of the protease inhibitors, aprotinin, pepstatin and leupeptin. Cells were lysed by 15 sec sonication, followed by a 1-min incubation at 65° C. Western blatting Twenty microliters of samples were separated on 12.5% SDS- PAGE. Antibodies against cdk4, cyelin D1, Rb and (i-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-PARP C-2-10 was obtained from Biomol. All antibodies were used at a concentration of 1:1.000 and immunoteactivity was demonstrated by ECL or color reaction, as described by the manufacnuer's protocol (Kirkegaatd and Perry, Gaithersburg, MD). liptd extraction . Cells were seeded at a density of 2 X 10s cells/ml as 25-m1 cultures. At the time points indicated, cells were harvested and transferred to screw top tubes with Teflon caps, and resuspended in 3 3 ml chlomform:methanol (1:2, v/v). The phases were separated by the addition of I ml chloroform and 1 ml 2% acetic acid. The bottom organic phase was ttansfened to a new tube and the aqueous phase was re-extracted with chloroform. The combined organic phases were dried, dissolved in 100 pl chloroform: methanol (95:5, v/v) and smred at -20°C. The DAG kinase assay was performed within 48 htr. Quantification of ceramrde and DAG using the DAG kinase assay Twenty microliters of each extracted lipid sample was dried in speed vac and dissolved in 7.5% octyl-S-glucoside, 5 mM cardio- lipin, 1 mM Detapac (pH 6.0) and sonicated 15 seo. After incubation at room temperature for 15 min, 20 µl 2X reaction buffer [100 mM 4-(2-hydroxyethyl)-1-pipemzinee0tane sulfonic acid (pH 7.0), 100 mM LiCl, 25 mM MgC1,, 2 mM EGTA, 2 mM DTTJ and 20 p1 DAG kinase solution (5 pg membranes in I mM Detapac, 10 mM imidazole) was added. Following the addition of V2P-y]ATP, samples were incubated at 30°C in a shaking water bath. After 1 hr, the reaction was stopped with the addition of 3 ml cliloroform:methanol (1:2). After the addition of 0.7 ml 1% HCl the phases were separated with 1 ml chloroform and I ml 1% HCI. The aqueous layer was aspirated and the organic phase was washed 2X with 1%HCI. For separation on TLC plates, 1 ml of the kinase assay mixture was dried and redissolved in 75 pl chloroform:methanol (95:5). Samples were applied to TLC plates and developed with chloroform: methanol:acetic acid (65:15:5). Spots corresponding to ceramide and DAG were removed and counted by liquid scintillation spectrometry. Counts were normalized to total phospholipids applied. Altetnatively, phosphorimager analysis was used to quan- tify the levels of ceramide and DAG, with comparable results. RESULTS Control of proteins involved in Gt/S transition by RA and 4HPR Expression of cyclin DI and cdk4. To investigate the mecha- nisms responsible for the partial arrest of cell cycling in RA- and 4HPR-treated cells, we examined changes in the levels of several key G,/S regulatory proteins. Cyclin Dl showed a 40% reduction after 24 hr of treatment with RA (corresponding to one doubling time); the decrease was sustained for 72 hr (Fig. la). In 4HPR- Fl treated cells, inhibition of cyclin D I expression was 30% at 24 hr, 75% at 48 hr and 50% at 72 hr. Since changes in cyctin DI may correspondingly affect the activity of cdk4, we next determined if there were concomitant changes in cdk4 expression. Results in F'rgure lb show that treatment with either retinoid altered the level of expression of this protein. By the first cell doubling of 24 hr thete was a significant 75% decrease in cdk4 in response to RA or 4HPR. However, by 48 hr, the cdk4 levels in 4HPR-treated cells returned to the control values. In the case of RA, no difference in cdk4 can be observed between control and treated cells at 72 hr. Hyperphosphorylation of Rb. To determine if the observed changes in cyclin D1 and cdk4 also affected the state of phosphory- lation of the retinoblastoma protein, changes in Rb and pRb were measured over a 3-day period. A decrease in phosphorylated Rb was evident by 24 hr and became more pronounced at 72 hr, in response to RA. Overall, 4HPR-heated cells showed a more significant reduction in pRb at all time points assayed (Fig. 2). Induction of apoptosis by 4HPR Processing of poly(ADP-ribose) polymerase. Previously we demonstrated that the 4HPR-induced apoptosis in the HL-60 cells occurred in parallel with the degradation of actin to a 15-kDa pmduct, suggesting that an ICE-like protease(s) may be involved in the mechanisN of 4HPR (DiPietrantonio et al., 1996). To elucidate further the role of pmteaces in the context of induction of apoptosis, cleavage of PARP, known to be an early event in apoptosis (Kaufmann et al., 1993), was evaluated. A 12-hr ueatment with F2

: UC (Cancer) - PAOE: 4 SESS: 9 OUTPUT: `Tue Ju¢ 23 23;59:48 1998 LtY 1 ssrijcjfijcjxxps/186k15 I 4 DUMRAN7ONIO SfAL A 6 h 12h 48 h 1 2 3 4 5 6 7 8 9 113K 89K -~ _ 0 500 400 6 12 24 Time (hours) ~ Control ®3µMRA 3 µM 4-HPR 48 b7oune 3-Gleavages of PARP to an 89Da fragment in response to 4APR treatment (a): Representative Western blot of PARP expression in unteeated (lanes 1,4, and'n, RA treated (lanes 2,5, and 8), and 4HPR treated (lanes 3,6, and 9) HL-60 cells. (b)Quanti5ceflon of the 89Da PARP fragment was identical to that described in the legend of Figure 1. Results are avemged from 3 separate experiments. 4HPR was accompanied by a 2.8-fold increase in the appeatance of control or R/,:keated cells. To ascertain further the involvement of 3 the 89-kDa form of PARP (Fig. 3a). Maximum processing of PARP ICE-like ptoteases in the 4HPR.elicited cleavage of PARP, the occurred at 24 hr (Fig. 3b). Cleavage of PARP was not observed in CPP32 inhibitor DEVD and the ICE inhibitor YVAD were

,1/ApME: IIC (GSneer) - PAGE:' 5 SESS: 9'OUTPUT: Tue JUn'23 23:59:48 1998 c$/lissffjcjrijcjxxps/186k15 I PIF3OlAOPtC IFFYL'IS OF4ttPR IN HUMAN tPUt¢MIA CMT iS Ihcnxe 4-Inhibition of PARP processing by the CPP32 inhibitor, DEVD. Quantification of PARP processing (as described in Fig. 3) fmm oells pretoeated overnight with DEVD or YVAD prior to the addition of 3pM 4HPR - separately tested for ability to inhibit the specific clGavage of PARP. Overnight pretreatment with 0.5-5.0 pM DEVD but not YVAD significantly reduced the specific processing of PARP (Fig. 4). Taken as a whole, these results suggest thirt the 4HPR-induced apoptosis probably involves an early activation of the protease CPP32, which in tum effects the cleavage of PARP to form the 89-kDa truncated product Analysis of DNA in 4fIPR-treated cells.lldditional evidence that HL60 cells treated with 4HPR undergo apoptosis came from 2 other experimental approaches. When DNA extracted from control and tre.ated cells was analyzed by agarose gel electtophoresis, DNA from 4HPR-treated and not control cells revealed the presence of DNA ladders that are characteristically associated with apoptotic cells (Fig. 5a). Apoptosis was also demonstrated immunohisto- chemically using an assay in which the relative abundance of the free 3'-ends in the DNA was measured in situ, based on the intensity of immunoperoxidase staining of digoxigenin nucleotides incorporated into the DNA. Results of such a study show that the fraction of cells undergoing apoptosis increased 5-fold in 4HPR- treated cells (Fig. 5b). Changes in ceranude and DAG levels in RA- or 4HPR-treated cells To obtain information on the signaling molecules that may trigger activation of CPP32, we investigated the changes in the levels of the lipid second messengers ceramide and DAG. Using the DAG Idnase assay (see Material and Methods), we determined the effect of RA and 4HPR on ceramide and DAG levels. An elevation in endogenous ceramide was seen as early as 3 hr in response to 4HPR treatment This 3-fold inctease was transient and not observed at earlier or later time points (Fig. 6a). In contrast to 4HPR there was no increase in the levels of ceramide following RA treatment, even after prolonged exposure for 24 hr. N.ither retinoid effectrd changes in the lcvels of DAG at any of the time points tested (Table I). We also determined the levels of these 2 5 lipid messengers in the breast cancer MCF-7 cells, which has been suggested to respond to either RA or 4HPR by undergoing apoptosis (Kazmi et al., 1996). A 3-hr treatment with either RA or 4HPR caused a significant increase in ceramide; this elevation was sustained from 3 to 6 hr in 4I-IPR-beated cells (Fig. 6b). There was no significant change in the levels of DAG in MCF-7 cells treated with either retinoid (data not shown). Studies with fumonisin B,, CPP32 and ICE inhibitors The studies described above show that induction of apoptosis by 4HPR occurs simultaneously with elevation in cetamide and activation of CPP32. To determine the temporal sequence in the change of these 2 biochemical parameters, following treatment with 4HPR, we studied the effects of FB„ an inhibitor of ceranilde synthase, and that of CPP32 and ICE inhibitors. Simultaneous treatment of cells with 4HPR and 25-100 µM FBI resulted in reduction in ceramide, accompanied by a corresponding decrease in the extent of PARP cleavage (Fig. 7ab), and a dose-dependent F7 suppression of apoptosis (Fig. 7c). However, pretreatment with the CPP32 or ICE inhibitors DEVD or YVAD, respectively, had no effect on the 4HPR-induced increase in ceramide (Fig. 7r!). Induction of apoptosis, on the other hand, was inhibited by 5 pM DEVD, an inhibitor of CPP32, and not by the same concentration of YVAD, an ICE inhibitor. These results suggest that elevation in ceramide occurs prior to the activation of CPP32. DISCUSSION Previous studies from our laboratory have shown that both RA and the synthetic retinoid 4HPR reduced HL-60 cell growth and arrested the HL-6t1 cell cycling at the G, phase (DiPietrantonio et al., 1996). However, 4HPR, in contrast to RA, also induces apoptosis in the HL60 cells. Details of how the 4HPR acts to effect apoptosis and other changes in the HL60 cells, and the signaling events it may elicit an: not characterized. Based on the above results and considerations, our first aim was to attempt to gain insights into the mechanism by which the target HG60 ce0s are adversely affected by RA and 4HPR. Toward that end, we performed experiments designed to test whether the observed G,to-S checkpoint dismption by RA and 4HPR occurs through a common mechanism. Since previous studies suggest that modulation of the stare of phosphorylation of the retinoblastoma protein Rb, by the edk4:cyclin DI complex, plays a key role in regulating this transition (Paggi et aL, 1996; Riley et a!. 1994), we determined changes in the expression of cyclin Dl, cdk4 and pRb. A reduction in the state of phosphorylation of Rb was observed, in both RA- and 4HPR-treated cells, which conelated with the suppressed expression of cyclin DI and cdk4 (Figs. 1, 2). The magnitude of these decreases appears to be sufficiently large as to compromise the phosphorylation of Rb. This in turn would result in an increase in the relative abundance of unphosphorylated Rb, which, by binding to the transcription factor E2F would restrict transcription of genes critical to the Gi-to-S progression. These changes in part could account for the cell cycle effects seen in RA- and 4HPR-tteated HL-60 cells. A second objective of the present studies was to probe the mechanism of induction of apoptosis by 4HPR. Apoptosis, or programmed cell death, is a multiphasic cellular ptogram involving numerous stage-specific genetic/morphologicaVbiochemical changes. Previously, we showed that 4HPR induces apoptosis in the HIr60 cells, as evidenced by the appearance of an additional "sub-Go " peak in flow cymmetric analysis, and the presence of a l5-kDa actin,iragment in Western blot analysis (DiPietmntonio et aL, 1996).>To validate these observations further, we assessed apoptosis in the present study by 3 additional approaches. First, we

/ DC (Cancer) - PAGE: 6 SFSS: 9 OUTPUT: 'ISie Jun 23 23:59:48 1998 V l/li's~sltjcj( jcj xxps/ 186k 15 6 A M12 © c~. ~ C O U ~ 'eY FYGOae 5-Induction of apoptosis in 411PR treated Hl.tells. (a): Agarose gel electrophoresis of DNA extracted from control (lane 1) or cells treated with 4HPR (lane 2) for 24 hr. (b): Quantification of cells undergoing apoprosis as measured by the Apotag assay (see Material and Methods). ~~ TABLE 1-CHANGPS IN DIACYLGLYCEROL (DAG) LEVELS IN t{l.6a CELLS TRF.ATED R7TH RA OR 4HPRI . Eane (havs/ 1 3 24 Control 100 100 100 3µMRA 129St41.1 134.9:t 60.7 110.3!31.9 3 pM 4-HPR 111.8 ± 65.8 168.6 ~ 35.5 88.5 t 49.4 'Results are a mean of 2 independent experiments performed in duplicate. assessed the cleavage of PARP, the DNA repair enzyme whose processing from an intact 113-kDa to a truncated 89-kDa form by ICI<like proteases is commonly accepted as an early biochemical index of apoptosis (Kaufmann et a1, 1993; Shah et at, 1996). These studies revealed the appearance of a processed 89-kDa PARP, following a 12- to 24br tteatment, in 4HPR- and not RA-treated or control cells (Fig. 3). PARP processing is effectively blocked with CPP32 and not ICE inhibitors (Fig. 4), strongly supporting the contention that CPP32 is activated in 4HPR-ueated cells to result in the processing of PARP. A second approach to evaluate 4HPR-induced apoptosis is the analysis of DNA isolated from control, RA- and 4HPR-neated cells. The appearance af DNA ladders, which presumably is generated by cleavage of DNA at the intemucleosomal regions, is only evident in 4HPR-treat<d celis (Fig. 5a). Finally we confirmed the induction of apoptosis by 411PR using an immunohistochemical assay in which the relative abun- dance of free 3'-ends in the DNA was quantified by labeling with the terminal deoxynucleotidyl tmnsfemse (F1&,jb). To probe the nature of molecules signaling the activation of , CPP32, we assessed changes in 2 lipid messengers (ceretnide and DAG). Ceramide, a product of spbingomyelin hydrolysis, has been proposed as an intracellular signaling molecule impm{9nt for mediating antiproliferative and apoptotic events (Hannun, 1994: Obeid and Hunnan, 1995; Spiegel et al., 1996). For example, the addition of Fas and TNF-a, both of which result in apoptosis, D@ETRAN70NIO6PAL increases ceramide levels (Cifone et aL, 1994; Dbaido et al., 1993). There am currently no reports linldng the generation of cetamide with the mechanism of 4HPR. Our present results showed an early increase in the levels of endogenous ceramide in response to 4HPR treatment (Fig. 6a). The same significant increases in ceramide levels were observed using the 413PR-treated MCF-7 breast cancer (Fig. 6b) and the JCA-1 prostate cancer cells (data not shown), preceding the induction of apoptosis. Additional evidence support- ing the involvement of ceramide in the 4HPR-induced apoptosis came from studies with the ceramide synthase inhibitor FB, which, when added simultaneously as 4HPR, effectively blocked the rise in ceramide, as well as processing of PARP, and induction of apoptosis (Fig. 7a-c). Collectively, these data support the possibility that ceramide is a key molecule in the signaling cascade initiated by 4HPR. In connast to the observed increase in ceramide levels, DAG, which, in principle, could play a role in the mechanism of action of 4HPR by activating protein kinase C, showed no significant change in its levels in response to either retinoid (Table 1). Thus it is unlikely that DAG is involved in the mechanism of action of RA or 4HPR in HL-60 cells. The exact manner in which cemmide generation may be critically linked to the induction of apoptosis by 4-HPR is unknown. Equally unknown is the manner by which transient ceramide increase leads to CPP32 activation, the deployment of CPP32 following activation, and the nature and properties of events downstream of CPP32. Possible downstream effects of ceramide include a ceramide-activated protein phosphatase (CAPP), a proline- directed protein kinase (PDPK) and the transcriptional activator NF-KB (Dobrowsky et a1., 1993; Joseph et al., 1993: Liu et al., 1994; Yang et af., 1993). ~ ACKNOWIEDGF.IvffiNTS This tesearch was supported by grants from the Vivian Wu-Au Memorial Cancer Research Fund and the Philip Morris Co. to 7MW.

AME: UC (Cancer) - PAGE: 7 SESS: 9 OUTPUT: Tuc Jun 23 23:59:48 1998 /Uss/ijcjfijcjxxpsl186k15 A 400 0 0 Control ® 3µMRA :`.'. ~ 3 µM 4HPR I B 200 ~ ~ ~ ~ ,.. , 100 ... o p8y ~ v y d u O ... ~ ~ b0 G a L U ~ Conwl ® 3µMRA 3 µM 4HPR 1 3 Time (hours) 3 - ~ Time (hours) 24 6 FIGURE 6-'IYmsient elevatioa of ceremide in mtinald treated HL-60 and MCF-7 celis. Results a`e avera¢ed from 2 ezneriments. cach

/AME: 1JC (Cencer)- PAGE: 8 SESS: 9 OU1'P[PC: Tue Jun 23 23:S:48 1998 " '8/lisslijcjfijcjxzpsl186k15 i A 250 ~ d > = 200 b- e ~ 0 150 V V V W .90 100 ~ 50 U 0 3 µM 4-HPR - + - Fumonisin Bl (µM) - - 25 0 50 + 25 N 60 0 20 0 + 50 f f f 3µ M 4-HPR + + + + Fum o nisin 25 50 100 Bl ( µ M) D E 200 8 0 ~ 7 0 4 I v 150 m 6 0 f s I I ~ 5 0 ~ L g 4 0 ~ 100 \ x.° 3 0 v~ ° ~ 50 W ° ~ 2 0 E 1 0 E .. 0 0 3 µM 4-HPR + + + + 3 µM 4 - HPR + + + + ' DEVD(µM) 0.5 5.0 DEVD ( µM) 0.5 5.0 - YVAD (FLM) 5.0 YVAD ( µ1q) 5.0 113K 89K 3 µM 4-HPR - + + + + Fumon(siob - - 25 50 100 Bl (µM) FtGUSe 7- Effects of FBI on ceremide elevation. PARP peaccgsing. and apoptosis, following 4HPR tteatment (a): the levels of ceramide were detamined, as descdbed in the legend of Figure 4. (b): '17u peteent of cetls undergoing apoptosis (c): Western blot analysis of PARP ezpression.

#AME: U'C; (Cancer) - PAGE: 9 SESS: 9 OUTPUT: Tue Jun 23 23:59:48 1998 i-° U)issr'c'fi c'xx s1186k15 PI.E1oTBOPIC F.E7F.CIS OF 4HPR IN HUMAN LEUKEMIA CELLS REFERENCES BAUtANt. K, BYnu, D.M, Cooa, H.W and Rnlowne, ND., Effect of fumonisin B, on phosphoethenolamine biosynthesis in Chinese hamster ovary cells. Bioehim biophyx Acm, 1304,190-196 (1996). CRAN, LN., ZIUNC, S., Stuo, J.. WAnm., R. TNOSnsoN. E.A. and CNAN. TS., N-(4-hydroxyphenyl)retinamide induces apoptosis in T lymphoma and T lymphoblastoid leukemia cells. Jeukemia Lymphoma, 25, 271-280 (1997). CtPom~, M.G., De MARU, R, Roxcuou, E, Rteeo, MR, AwstA, M, LANmt LL, SANtona, A and 1tsn, R, Apoptotic signaling through CD95 (Fas/Apo-l) activates an acidic sphingomyelinase. J. exp. Med, 177, 1547-1552 (1994). 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