See related Commentary on page iv

The Novel Synthetic Oleanane Triterpenoid CDDO (2-cyano-3,12-dioxoolean-1, 9-dien-28-oic acid) Induces Apoptosis inMycosis Fungoides/Sezary Syndrome Cells

Chunlei Zhang,� Xiao Ni,� Marina Konopleva,w Michael Andreeff,w and Madeleine Duvic�

Departments of �Dermatology and wBlood & Marrow Transplantation, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA

The novel synthetic oleanane triterpenoid CDDO (2-cyano-3, 12-dioxoolean-1, 9-dien-28-oic acid) can serve as a

ligand for the peroxisome proliferator activator receptor-c (PPAR-c) and has been shown to inhibit cell proliferation,

and to induce differentiation and apoptosis in tumor cell lines. Bexarotene is an RXR-selective retinoid that can

induce apoptosis of mycosis fungoides (MF) and Sezary syndrome (SS) cells. Since the PPAR-c and RXR receptors

can form heterodimers, we studied the effects of CDDO and its synergism with bexarotene on apoptosis in MF/SS

cell lines (MJ, Hut78, and HH) and freshly isolated peripheral blood lymphocytes (PBL) from SS patients with

circulating atypical T cells (CD4þCD26�). CDDO treatment at 1–5 lM for 48 h caused a concentration-dependent

apoptosis in three MF/SS cell lines and patients’ PBL compared to vehicle controls. Bexarotene augmented CDDO-

induced apoptosis in these cells. PPAR-c was expressed but decreased by 47% in MJ, 42% in Hut78, and 77% in HH

cells following CDDO treatment. The anti-apoptotic protein bcl-xL, but not bcl-2, was decreased by 69% in MJ, 31%

in Hut78, and 59% in HH cells and caspase-3 was activated following CDDO treatment. Interestingly, the PPAR-cantagonist T007 did not block CDDO-induced apoptosis, and the more potent PPAR-c agonist rosiglitazone

required much higher concentrations (450 lM) than CDDO to induce apoptosis in MF/SS cells. In summary, CDDO

induces apoptosis that is further enhanced by bexarotene and decreases the PPAR-c and bcl-xL proteins in MF/SS

cells. CDDO’s effects on MF/SS cells may be at least partly mediated through a PPAR-c-independent mechanism.

Our findings suggest the rationale for further investigation of the clinical potential of CDDO, either alone or in

combination with bexarotene for MF/SS patients.

Key words: bcl-xL/mycosis fungoides/PPAR-g/RXR/Sezary syndromeJ Invest Dermatol 123:380 –387, 2004

Cutaneous T cell lymphomas (CTCL) are a group oflymphoproliferative disorders characterized by localizationof malignant T lymphocytes to the skin at presentation.Mycosis fungoides (MF), the most common and indolentform of CTCL, is characterized by patches, plaques ortumors containing epidermotropic CD4þCD45ROþ helper/memory T cells. Prognosis in tumor-stage MF is poor, with amedian survival of less than 40 months. Sezary syndrome(SS) is the leukemic form of CTCL, which presents witherythroderma and is characterized by atypical cells in theperipheral blood and by a peripheral blood T cell clone.Sezary syndrome is an aggressive disease with a poorprognosis and a median survival of only 30 months (Duvicand Cather, 2000; Siegel et al, 2000). Early MF can often becontrolled with skin directed or biological response modi-fiers including topical steroids or retinoids, phototherapy,topical chemotherapy, interferon, cytokines, extracorporealphotopheresis, or total skin electron beam irradiation

(Apisarnthanarax and Duvic, 2001). Although partial andcomplete remissions are achieved, subsequent relapses arecommon. Alternative or complementary therapies, espe-cially for advanced MF/SS, are needed. Dysregulatedapoptosis of skin-homing memory T cells is involved inpathogenesis and therapeutic implications of MF/SS (Ka-cinski and Flick, 2001; Meech et al, 2001). Pharmacologicalmodulation of apoptosis may provide new therapeuticapproaches for this disease.

The novel synthetic oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1, 9-dien-28-oic acid or CDDO is a multi-functional molecule with promising clinical potential as achemopreventive agent and as a therapeutic agent for thetreatment of cancer (Suh et al, 1999). In vitro studies haveshown that CDDO induces cell differentiation, growthinhibition, and apoptosis in human leukemia (Ito et al,2000; Pedersen et al, 2002), osteosarcoma (Ito et al, 2001)and breast cancer (Lapillonne et al, 2003) cell lines. Thestructure of CDDO resembles a steroid hormone and otherisoprenoids. More recent studies have suggested thatCDDO is a ligand for the peroxisome proliferator activatorreceptor-g (PPAR-g) (Wang et al, 2000).

PPAR-g is a member of the nuclear hormone receptorsuperfamily and thus acts as a ligand-dependent transcrip-tion factor controlling cellular differentiation, proliferation

Abbreviations: CDDO, 2-cyano-3, 12-dioxoolean-1, 9-dien-28-oicacid; CTCL, cutaneous T-cell lymphomas; 15d-PGJ2, 15-deoxy-D12,14-Prostaglandin J2; MF, mycosis fungoides; PARP, poly (ADP-Ribose) polymerase; PBL, peripheral blood lymphocytes; PI,propidium iodide; PPAR-g, peroxisome proliferator activatorreceptor-g; SS, Sezary syndrome

Copyright r 2004 by The Society for Investigative Dermatology, Inc.

380

and apoptosis. After ligand binding, PPAR-g forms aheterodimer with the retinoid X receptor (RXR) (Mangelsdorfet al, 1995). We have previously demonstrated that an RXRligand, bexarotene, is effective in reducing the cutaneousmanifestations of MF/SS in both early and late stagepatients (Duvic et al, 2001). Bexarotene can also induceapoptosis of MF/SS cell lines in vitro (Zhang et al, 2002).Thus, induction of apoptosis may be one mechanism ofbexarotene action in the treatment of MF/SS patients.

In this study, we determined the independent andpotentially synergistic effects of CDDO and other PPAR-gligands and bexarotene on apoptosis induction as well asgene expression in malignant MF/SS cell lines and freshlyisolated peripheral blood lymphocytes (PBL) from Sezarysyndrome patients.

Results

Effect of CDDO on growth inhibition in MF/SS cells Todetermine whether CDDO inhibits MF/SS cell growth, MJ,Hut78, and HH cells were treated with or without 1, 2, and5 mM CDDO for 48 h and their viability was evaluated byCellTiter 96 AQ One Solution Cell Proliferation Assay. As theconcentration of CDDO increased from 1 to 5 mM over 48 h,cell growth inhibition was significantly increased from 21%to 72% (MJ), 23% to 70% (Hut78), and 12% to 59% (HH) ina concentration-dependent manner compared with vehiclecontrols (n¼3; po0.05), respectively (Fig 1). These resultsdemonstrated that CDDO is a potent inhibitor of MF/SS cellgrowth.

Effect of CDDO on cell cycle and apoptosis in MF/SScells To determine whether growth inhibition of CDDO isdue to cell-cycle arrest and/or apoptosis in MF/SS cells,MJ, Hut78, and HH cell lines were treated with or without 1,2, and 5 mM CDDO for 48 h. As the concentration of CDDOincreased from 1 to 5 mM, the number of cells with sub-G1

populations increased from 15% to 58% (MJ), 10% to 56%

(Hut78), and 12% to 47% (HH) in a concentration-depen-dent manner compared to vehicle control (n¼3; po0.05).Increased numbers of cells in the sub-G1 population wereaccompanied by a loss of cells in the G1, S, and G2/Mphases (Fig 2). It has been reported that cells with thesefeatures are those dying of apoptosis (Nicoletti et al, 1991).This indicated that all three cell lines undergo apoptosisrather than cell-cycle arrest under CDDO treatment.

In parallel to the cell-cycle analysis, we investigated theeffect of CDDO on annexin V-binding, an early hallmark ofapoptosis, in MF/SS cells (Vermes et al, 1995). As theconcentration of CDDO increased from 1 to 5 mM for 48 h,the number of cells stained for annexin V increased from11% to 65% (MJ), 11% to 73% (Hut78), and 6% to 69%(HH) in a concentration-dependent manner compared tovehicle control (n¼ 3; po0.05). Similar results were alsoseen in freshly isolated PBL from Sezary syndrome patientswith circulating atypical T cells (Fig 3). These results wereconsistent with analysis of cell-cycle distributions frompropidium iodide (PI) staining, further confirming that MF/SScells undergo apoptosis following CDDO treatment.

Synergistic effect of CDDO and bexarotene on apopto-sis induction in MF/SS cells To determine whether CDDOand bexarotene can synergize to induce apoptosis in MF/SS cells, three cell lines and patients’ PBL were treated with1 mM CDDO, 10 mM bexarotene, or their combination for 48h. CDDO treatment alone increased the sub-G1 populationby 14% and annexin V binding by 20% compared withvehicle control. Bexarotene alone increased sub-G1 by only2% and annexin V binding by 3% over vehicle control. Thecombination of CDDO and bexarotene increased thepercentage of cells in sub-G1 populations by 37% andannexin V binding by 49% in the HH cell line (Fig 4). Similarresults were also seen in other cell lines and patients’ PBL(not shown). The above observation possibly suggests thata combination of CDDO and bexarotene has synergisticpro-apoptotic effects in MF/SS cells.

Effect of CDDO on expression of apoptosis-associatedproteins in MF/SS cells To understand the mechanism ofCDDO-induced apoptosis, we examined the expressionof nuclear receptor PPAR-g, death receptor Fas/FasL, anti-apoptotic proteins bcl-2 and bcl-xL, and caspase-3 andpoly (ADP-Ribose) polymerase (PARP) proteins in three MF/SS cell lines (MJ, Hut78, and HH). Western blotting showedthat PPAR-g protein was expressed and was decreased by47% in MJ, 42% in Hut78, and 77% in HH cells followingtreatment with 2 mM CDDO for 48 h. Expression of Fas/FasLproteins was not changed following CDDO treatment. Bcl-xL, but not bcl-2 protein, was decreased by 69% in MJ,31% in Hut78, and 59% in HH cells following CDDOtreatment. CDDO treatment was also associated withcaspase-3 activation and PARP cleavage as determinedby the decrease of procaspase-3 and appearance of 85 kDafragments in all three MF/SS cell lines (Fig 5).

Effect of other PPAR-c ligands and antagonist onapoptosis in MF/SS cells To determine whether otherPPAR-g ligands also induce apoptosis in MF/SS cells, threecell lines were treated with 15-deoxy-D12,14-Prostaglandin

Figure 1CDDO treatment inhibits cell growth of MF/SS cells. Cells (5 � 104)were aliquoted into a 96-well culture plate and grown in medium with orwithout 1, 2, and 5 mM CDDO for 48 h. Cell viability was determined bycomparing untreated control (DMSO) to treated groups with theCellTiter 96 AQueous One Solution Cell Proliferation Assay performedin triplicate. Each point represented mean � SD of triplicate determi-nations. �Significantly different from DMSO control values (n¼3;po0.05).

CDDO INDUCES APOPTOSIS IN MF/SS CELLS 381123 : 2 AUGUST 2004

J2 (15d-PGJ2) (1, 10, and 50 mM) and rosiglitazone (1, 10,50, 100, and 200 mM) for 48 h. As the concentration of 15d-PGJ2 increased from 10 to 50 mM, the number of HH cellswith annexin V binding increased from 67% to 94% ina concentration-dependent manner over vehicle control(Fig 6A). Rosiglitazone required much higher concentrations(50–200 mM) than CDDO to induce annexin V binding by3%–33% in the HH cell line (Fig 6B). Similar results werealso seen in MJ and Hut78 cell lines (not shown). Moreover,T007, a PPAR-g-specific antagonist, did not block theCDDO-induced apoptosis in three cell lines (Fig 7).

Discussion

Apoptosis is a genetically regulated form of cell deathcharacterized by specific morphologic and biochemicalchanges that differ from necrosis (Kerr et al, 1972).Apoptosis plays an important role in embryogenesis, aging,and many diseases including cancer (Webb et al, 1997). Theanti-tumor actions of many chemotherapeutic agents havebeen attributed to induction of apoptosis (Hickman, 1992).In MF/SS, effective treatments such as phototherapy (Yoo

et al, 1996), photopheresis (Bladon and Taylor, 1999) andrexinoids (Zhang et al, 2002) work through induction of T cellapoptosis. In this study, we show for the first time thatCDDO causes apoptosis of MF/SS cells in association withdownregulation of bcl-xL. PPAR-g expressed in MF/SS cellsis suppressed following CDDO treatment. Importantly,bexatotene augments CDDO-induced apoptosis in MF/SScells, suggesting synergism.

Apoptosis is a multistep process and increasing numbersof genes have been identified that are involved in the controlor execution of apoptosis (Synderman, 2001). Caspasesplay a crucial role in apoptosis. Among the 14 knownmembers of interleukin-1-converting enzyme family ofproteases, caspase-3 has been shown to be a key compo-nent of the apoptotic machinery (Nunez et al, 1998).Caspase-3 is activated in apoptotic cells and cleavesseveral cellular proteins, including PARP. The cleavage ofPARP is used as a hallmark of apoptosis by various anti-tumor agents (Duriez and Shah, 1997). In this study, weshow that CDDO treatment triggers apoptosis of three MF/SS cell lines in association with caspase-3 activation andPARP cleavage, implying that activation of caspase-3 isinvolved in CDDO-induced apoptosis.

Figure2CDDO treatment increases sub-G1 po-pulation in MF/SS cells. Cells (1 � 106)were treated with or without 1, 2, and 5mM CDDO for 48 h. Cell-cycle distributionwas determined by DNA content analysiswith PI staining and flow cytometry. (A)Each point represented the percent ofcell-cycle distributions with different DNAcontent. (B) Histograms for cell cycle werefrom analysis of MJ, Hut78, and HH cellstreated with 2 mM CDDO for 48 h. Resultswere representative of one of three inde-pendent experiments.

382 ZHANG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Fas/FasL pair plays a critical role in regulating the life anddeath of T lymphocytes (Varadachary and Salgame, 1998).Fas is expressed on activated lymphocytes, but itsexpression is decreased or lost in malignant MF/SS cells(Dereure et al, 2000; Zoi-Toli et al, 2000; Zhang et al, 2002).Impaired Fas/FasL apoptotic pathway may contribute tothe pathogenesis of MF/SS and could be manipulatedfor therapeutic application. In this study, we found thatCDDO did not change the expression of Fas/FasL proteinsin MF/SS cells, suggesting that an apoptotic pathwayindependent of Fas/FasL interactions may be involved inCDDO-induced apoptosis.

The anti-apoptotic proteins, bcl-2 and bcl-xL, have beendemonstrated to play an important role in the regulation ofapoptosis and to inhibit the induction of apoptosis inlymphocytes by a variety of signals (Reed, 1998). Expres-sion of bcl-2 and bcl-xL is found in MF/SS cells and ishypothersized to increase survival and resistance of MF/SScells against radiotherapy and extracorporeal photoche-motherapy (Kanavaros et al, 1994; Dummer et al, 1995;Osella-Aate et al, 2001; Zhang et al, 2002, 2003). Recentstudies have shown that bcl-2 and bcl-xL play a critical rolein controlling the activation of caspases by regulatingrelease of cytochrome c from mitochondria (Yang et al,1997; Reed, 1998). This indicates that both proteinsfunction upstream of the caspase activation step in the celldeath pathway. In this study, we show that CDDO treatmentdecreased the level of bcl-xL but not bcl-2 in all three celllines, suggesting that bcl-xL rather than bcl-2 is involved inCDDO-induced apoptosis of MF/SS cells.

PPAR-RXR heterodimers can be activated by PPAR orRXR ligands (Forman et al, 1997; Nolte et al, 1998), andRXR-specific ligands markedly induce the binding of SRC-1to PPAR-RXR heterodimers (Westin et al, 1998). Assemblyof this complex results in a large increase in transcriptionalactivity. It has been shown that simultaneous activation ofboth receptors can yield maximal anti-diabetic activation invivo (Mukherjee et al, 1997). Combinations of the PPAR-gligands with the RXR ligands were shown to markedlydecrease cell growth and induce monocytic differentiationin myeloid leukemic HL-60 cells (Tontonoz et al, 1998).Therefore, it was logical to try to enhance the effects ofPPAR-g ligands by combining them with the novel syntheticRXR ligand bexarotene. In this study, we show for the first

time that PPAR-g is expressed and downregulated followingCDDO treatment in MF/SS cells. CDDO induced apoptosisof MF/SS cells, and bexarotene enhanced the CDDO-induced apoptosis. We previously reported that bexaroteneat clinically relevant concentrations (1–10 mM) requireslonger incubation (96 h) to induce apoptosis of MF/SScells. Furthermore, bexarotene at 24 and 48 h, prior tocausing apoptosis, induces some molecular events such asdownregulation of the expression of its cognate receptor,RXR-a, in MF/SS cells (Zhang et al, 2002). This may give us

Figure 3CDDO treatment increases annexin Vbinding to MF/SS cells. Cells (1 � 106)were treated with or without 1, 2, and 5mM CDDO for 48 h. Annexin V binding wascarried out with the Annexin V-FITCDetection Kit. (A) Each point representedthe percentage of both annexin Vþ PI�

and annexin VþPIþ cells. (B), annexin V/PI staining was analyzed from the Hut78cell line and one Sezary syndrome pa-tient’s PBL. The lower right quadrantshowed annexin Vþ PI� cells. The upperright quadrant showed annexin VþPIþ

cells. Results were representative of oneof three independent experiments.

CDDO INDUCES APOPTOSIS IN MF/SS CELLS 383123 : 2 AUGUST 2004

a clue to explain why bexarotene alone for 48 h did notinduce apoptosis but has the synergy with CDDO in thisstudy. Further experiments for cross talk between RXR andPPAR-g in MF/SS remain to be set up.

On the other hand, we tested other PPAR-g ligands 15d-PGJ2 and rosiglitazone in inducing apoptosis of MF/SScells. Like CDDO, 15d-PGJ2, a natural PPAR-g ligand(Forman et al, 1995), significantly induced apoptosis inMF/SS cells. In contrast, the well-characterized syntheticand high-affinity PPAR-g ligand, rosiglitazone (Lehmannet al, 1995), required much higher concentrations (450 mM)than CDDO to induce a lower level of apoptosis in MF/SScells. Moreover, the PPAR-g-specific antagonist T007 didnot block the CDDO-induced apoptosis in MF/SS cells. Ithas been known that CDDO and 15d-PGJ2 recruit the co-activator DRIP205 in myeloid leukemic cells, whereas othersynthetic potent PPAR-g ligands such as rosiglitazone donot have this activity (Kodera et al, 2000; Tsao et al, 2001).These findings suggest that CDDO-induced apoptosis is, atleast partially, mediated through PPAR-g-independent me-chanisms in MF/SS cells.

Indeed, CDDO has been reported to have other PPAR-g-independent activities, including suppression of induciblenitric-oxide synthase and cyclooxygenase-2 (Suh et al,1998; Honda et al, 2000), inhibition of NF-kB mediated geneexpression (Stadheim et al, 2002), and enhancement ofTGF-b/Smad signaling (Suh et al, 2003). Given the diversebiological activities of CDDO, we are therefore left with theconclusion that it is likely that another functional receptorsystem (or systems) beyond PPAR-g remain to be identifiedas CDDO target in MF/SS cells.

In conclusion, we demonstrated that CDDO inducesapoptosis of MF/SS cell lines and freshly isolated PBL fromSezary syndrome patients with circulating atypical T cells(CD4þCD26�). These events are associated with down-regulation of PPAR-g and bcl-xL, and activation of caspase-3. Bexarotene augments CDDO-induced apoptosis in MF/SS cells. Our findings provide a rationale for the clinical

potential of CDDO, either alone or in combination withbexarotene for MF/SS patients. Further experiments areneeded to confirm the results and clarify the moleculardetails of their proapoptotic effects for clinical development.

Materials and Methods

Reagents CDDO was manufactured under the NIH RAID Programunder good manufacturing practice conditions and kindly providedby Drs Edward A. Sausville and Michael B. Sporn. PPAR-g ligandsincluding rosiglitazone (BRL49653) and 15d-PGJ2 were purchasedfrom Cayman Chemical (Ann Arbor, Michigan). T0070907 (referredthereafter as T007), a selective PPAR-g antagonist (Lee et al, 2002),was synthesized and kindly provided by Dr Stephen H. Safe (TexasA&M University, College Station, Texas). CDDO, rosiglitazone, andT007 were dissolved in dimethyl sulfoxide (DMSO) to a stockconcentration of 10 mM and stored in �201C. Serial dilutions (1, 2,5, 10, 50, 100, and 200 mM) were freshly made in RPMI 1640.Bexarotene (LGD1069, 4-[1-(3, 5, 5, 8, 8-pentamethyl-5, 6, 7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid) was obtained fromLigand Pharmaceuticals Inc. (San Diego, California). A stocksolution of bexarotene at the concentration of 10 mM wasprepared in DMSO and stored at �201C in the dark.

Cells and cell culture Human CTCL cell lines MJ (G11), Hut78,and HH obtained from American Type Culture Collection (Rockville,Maryland) were derived from peripheral blood of patients with MF,SS, and non-MF/SS aggressive CTCL, respectively (Gootenberg etal, 1981; Popovic et al, 1983; Starkebaum et al, 1991). Samples ofperipheral blood were obtained for in vitro studies from three SSpatients with circulating atypical T cells (CD4þCD26�). Sampleswere obtained during routine diagnostic assessments afterinformed consent was obtained in accordance with regulationsand protocols sanctioned by the Human Subjects Committee ofMD Anderson. PBL from these patients were isolated byVacutainer CPT (Becton Dicknson, San Jose, California). Cellswere grown in RPMI 1640 medium (Sigma Chemical, St Louis,Missouri) supplemented with 10% heat-inactivated fetal bovineserum (HyClone Laboratories, Logan, Utah), 2 mM HEPES, and 1%penicillin–streptomycin in a humidified atmosphere with 5% CO2 at371C.

Figure4Synergistic effect of CDDO and bexar-otene on apoptosis induction in MF/SScells. HH cells (1 � 106) were treated with1 mM CDDO, 10 mM bexarotene, or theircombination for 48 h. Apoptosis wasmeasured by flow cytometry analysis ofsub-G1 and annexin V binding population.(A), histograms for cell cycle were ana-lyzed by PI staining and flow cytometry.(B), annexin V/PI staining was analyzed byflow cytometry. Results were representa-tive of one of three independent experi-ments.

384 ZHANG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Cell viability Cell viability was measured by CellTiter 96 AQ OneSolution Cell Proliferation Assay (Goodwin et al, 1995) according tothe manufacturer’s instructions (Promega, Madison, Wisconsin).Aliquots of 5 � 104 cells/well were distributed in 96-well micro-plates (Falcon, Franklin Lakes, New Jersey) in 100 mL of mediumand incubated with 1, 2, and 5 mM CDDO for 48 h, then 20 mL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-

phenyl)-2H-tetrazolium, inner salt was added to each well andincubated for an additional 4 h. The relative cell viability wasdetermined by ELISA with a 490 nm filter. Each experiment wasperformed in triplicate, and repeated a minimum of three times.

Flow cytometry analysis of cell cycle MJ, Hut78, and HH cells(1 � 106 cells) were incubated with or without 1, 2, and 5 mMCDDO for 48 h. Cells were collected, washed with cold phosphate-buffered saline (PBS), fixed in cold (�201C) 100% ethanol, treatedwith DNase-free RNase, and stained with 50 mg per mL PI.Distribution of the cell-cycle phase by different DNA content wasdetermined with a FACScan flow cytometer (Becton Dickinson,San Jose, California). For each sample, 10,000-gated events wereacquired. Analyses of cell-cycle distribution (including apoptosis:sub-G1) were performed using Modfit software (Becton Dickinson).

Annexin V binding staining The analysis of annexin V bindingwas carried out with an Annexin V-FITC Detection Kit I (PharMin-gen, San Diego, California) according to the manufacturer’sinstructions. Briefly, three cell lines (MJ, Hut78, and HH) andpatients’ PBL were incubated with or without CDDO (1, 2, and5 mM), bexarotene (10 mM), T007 (2 mM), 15d-PGJ2 (1, 10,and 50 mM), rosiglitazone (1, 10, 50, 100, and 200 mM), or theircombinations for 48 h. Cells were collected, washed twice withcold PBS, and centrifuged at 300 g for 5 min. Cells wereresuspended in 1 � binding buffer at a concentration of 1 � 106

cells per mL, 100 mL of the solution was transferred to a 5 mLculture tube, and 5 mL of annexin V-FITC and 5 mL of PI wereadded. Cells were gently vortexed, and incubated for 15 min atroom temperature in the dark. Finally, 400 mL of 1 � binding bufferwas added to each tube, and samples were analyzed by FACScanflow cytometer (Becton Dickinson). For each sample, 10,000 un-gated events were acquired. Annexin VþPI� cells represent theearly apoptotic populations. Annexin VþPIþ cells represent eitherlate apoptotic or secondary necrotic populations.

Isolation of cytoplasmic and nuclear extracts Cells (5 � 106)were washed with ice-cold PBS, harvested into 1 ml of PBS,pelleted in a 1.5 mL microcentrifuge tube, and suspended in 400mL of buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 1 mM EDTA,1 mM EGTA, 1 mM DTT, and 1 � protease inhibitor cocktail‘‘complete mini’’ (Roche, Indianapolis, Indiana)]. After a 20 minincubation on ice, the mixture was treated with a 24-G syringe forfive times and then centrifuged briefly to obtain the cytoplasmicsupernatant. The nuclear pellet was resuspended in 40–80 mL ofbuffer C [10 mM HEPES (pH 7.9), 10 mM KCl, 1 mM EDTA, 1 mMEGTA, 1 mM DTT, 10% glycerol, and 1 � protease inhibitorcocktail ‘‘complete mini’’ (Roche)] and incubated at 41C withshaking for 15 min. Protein concentrations were determined by theBradford dye-binding protein assay (Bio-Rad, Richmond, Califor-nia) using bovine serum albumin as a standard.

Western blot analysis Cytoplasmic (20 mg) or nuclear (10 mg)proteins were subjected to sodium dodecyl sulfate-polyacrylamidegel (8–12%) electrophoresis (SDS-PAGE) and transferred ontonitrocellulose membranes (Schleicher & Schuell, Dasse, Germany).The membranes were blocked in 3% powdered milk in TBST [50

Figure5Effect of CDDO on nuclear receptor and apoptosis-relatedproteins in MF/SS cells. Cytoplasmic (20 mg) or nuclear (10 mg)proteins from MJ, Hut78, and HH cells treated with 2 mM CDDO for 48 hwere fractionated by 8%–12% SDS-PAGE, transferred onto nitrocellu-lose membranes and subjected to the ECL detection analysis. Theequivalent loading of proteins in each well was confirmed by actin. Theprotein levels were quantified by AlphaEase 4.0 Imager. The amount ofPPAR-g and bcl-xL proteins from untreated controls was expressed as100%. Lanes 1 and 2, MJ; Lanes 3 and 4, Hut78; Lanes 5 and 6, HH;Lanes 1, 3, and 5, vehicle control (DMSO); Lanes 2, 4, and 6, 2 mMCDDO; Lane 7, positive control for Fas protein.

CDDO INDUCES APOPTOSIS IN MF/SS CELLS 385123 : 2 AUGUST 2004

mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween 20], incubated withprimary antibody overnight at 41C in 3% powdered milk in TBST,and washed extensively with TBST. They were incubated with1:5000 peroxidase-conjugated anti-mouse or anti-rabbit second-ary antibody (Jackson ImmunoResearch, West Grove, Pennsylva-nia) for 1 h at room temperature. Monoclonal mouse anti-Fas andanti-FasL were obtained from Transduction Laboratories (SanDiego, California). Monoclonal mouse anti-actin, anti-bcl-2, anti-PPAR-g, anti-caspase-3 and anti-PARP, and polyclonal rabbit anti-bcl-xL were obtained from Santa Cruz Biotechnology (Santa Cruz,California). Immunoreactive bands were visualized by enhancedchemiluminescence detection kit (Amersham, Buckinghamshire,UK). The equivalent loading of proteins in each well was confirmedby Ponceau staining and actin control. The protein levels werequantified by an AlphaEase 4.0 Imager (Alpha Innotech Co., SanLeandro, California).

Statistical analysis All experiments were performed in triplicateunless otherwise noted. Results were expressed as means � SD.

Statistical significance was evaluated by a two-tailed, paired,Student’s t test, and po0.05 was considered as significant.

We thank Wendy Schober-Ditmore for technical help in flow cytometry.We also thank Dr Parul Hazarika for her suggestions while writing thismanuscript.

This work was presented in part at the Society for InvestigativeDermatology Meeting, May 15–18, 2002, Los Angeles, CA. It wassupported in part by grants from National Institutes of Health (R21-CA74117 & K24-CA 86815; to M.D.), the M.D. Anderson Cancer CenterCore Cancer Grant (CA 16672), and the CTCL Patient Education Fund,Sherry L. Anderson CTCL Research Fund.

DOI: 10.1111/j.0022-202X.2004.23207.x

Manuscript received December 18, 2003; revised March 16, 2004;accepted for publication March 23, 2004

Address correspondence to: Madeleine Duvic, Department of Derma-tology—Box 434, The University of Texas MD Anderson Cancer Center,1515 Holcombe Blvd., Houston, TX 77030, USA. Email: mduvic@mdanderson.org

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Figure 6Effect of other PPAR-c ligands on apoptosis induction in MF/SS cells. HH cells were treated with (A) 15d-PGJ2 (1, 10, and 50 mM) and (B)rosiglitazone (1, 10, 50, 100, and 200 mM) for 48 h. Apoptosis was measured by flow cytometry analysis of annexin V and/or PI binding. Each valuerepresented the percentage of both annexin VþPI� and annexin VþPIþ cells. Results were representative of one of three independent experiments.

Figure 7Effect of the PPAR-c antagonist on CDDO-induced apoptosis inMF/SS cells. MF/SS cells were treated with CDDO (2 mM), T007 (2 mM)or their combination for 48 h. Apoptosis was measured by flowcytometry analysis of annexin V and/or PI binding. Each pointrepresented the percentage of both annexin VþPI� and annexinVþPIþ cells. Results were representative of one of three independentexperiments.

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