ICANCER RESEARCH57, 4916-4923. November 1, 19971

ABSTRACT

We have shown that cell cycle progression of human T-cell leukemiavirus type I (HTLV-I)-transformed T-cell lines was inhibited by 13-cisretinoic acid (13cRA). In the present study, we report that 13cRA inhibited proliferation and induced cell death of peripheral blood mononuclearcells obtained from four patients with acute adult T-cell leukemia but notof mitogen- or interleukin 2-activated peripheral blood mononuclear cellsfrom HTLV-I-negative healthy donors. Because HTLV-I-infected lymphocytes are susceptible to oxidative stress, we examined the role of theintracellular redox state in 13cRA-induced cell death using a HTLV-Ipositive T-cell line, ATL2, as a model. 13cRA induced apoptosis in ATL2

cells within 48 h in a dose-dependent manner. The ability of 13cRA toinduce apoptosis was more potent than that of all-lrans-retinoic acid.Apoptosis induction by 13cRA was significantly enhanced by buthiomnesulfoximine (BSO), which decreased the leveis of intracellular reducedglutathione, although 13cRA by itself did not alter them, suggesting thatintracellular reduced glutathione may modulate 13cRA-induced apoptosis. In addition, flow cytometric analysis revealed that 13cRA increasedintracellular peroxides in 24 h and that the addition of BSO furtherenhanced them. Although N-acetylcysteine had only a marginal effect,pretreatment with catalase markedly inhibited 13cRA-induced apoptosis.These results suggest that peroxide generation, i.e., oxidative stress, mayplay a crucial role in the induction of apoptosis by 13cRA and furtherdemonstrate that combined treatment with 13cRA and BSO inducesapoptosis of HTLV-I-positive lymphocytes even more potently.

INTRODUCTION

ATL,4 an aggressive and often fatal lymphoid malignancy, is eliologically associated with HTLV-I (1). However, the precise mecha

nism by which HTLV-I infection develops into leukemia remainspoorly understood, and ATh remains resistant to most conventionalchemotherapies. We have previously shown that 13cRA preferentiallyinhibits the proliferation of HTLV-I-transformed T-cell lines (2). RAs

are derivatives of vitamin A and are known to exert a wide variety ofeffects on vertebrate development, cellular differentiation, and prolif

eration (3—5).Whereas diverse effects of atRA or 9cRA are known tobe mediated by two nuclear receptors, the RAR (6, 7) and the RXR (8,

Received 4/2 1/97; accepted 9/3/97.The costs of publication of this article were defrayed in part by the payment of page

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

I Supported by a Grant-in-Aid for Scientific Research and Special Project Research in

Cancer Bioscience from the Ministry of Education, Science, Sports and Culture of Japanand by the Life Science Research Project of the Institute of Physical and ChemicalResearch.

2 Present address: Division of Cellular and Gene Therapies (HFM-5l8), Center for

Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD.3 To whom requests for reprints should be addressed, at Department of Biological

Responses, Institute for Virus Research, Kyoto University, 53 Kawaharacho, Shogoin,Sakyo-ku, Kyoto 606-01, Japan. Phone: 81-75-751-4024; Fax: 81-75-761-5766.

4 The abbreviations used are: AU, adult T-cell leukemia: HTLV-I, human T-cell

leukemia virus type I; RA, retinoic acid; l3cRA, 13-cis-RA; PBMC, peripheral bloodmononuclear cell; atRA, all-trans-RA; BSO, buthionine sulfoximine; GSH, reducedglutathione; RAR, RA receptor: RXR, retinoid X receptor; 9cRA, 9-cis-RA; TRX.thioredoxin; ROI, reactive oxygen intermediate; NAC, N-acetylcysteine; PHA, phytohemagglutinin; IL-2, interleukin 2; rlL-2, recombinant IL-2; AO, acridine orange; EB,ethidium bromide; P1, propidium iodide; Ab, antibody; DCFH, 2',7'-dichlorofluorescein.

9),bothofwhichcantransactivateseveralresponsivegenes(10),theexact signaling pathway of I3cRA is poorly understood. Because thebinding affinities of 13cRA to RAR or RXR are significantly weakcompared to those of atRA or 9cRA (1 1), it is suggested that 13cRAmay activate a signaling pathway distinct from these nuclearreceptors.

There is growing evidence that cell death and proliferation areregulated by a number of redox-related proteins such as TRX (12, 13),superoxide dismutase (14), glutathione peroxidase (15), catalase (16),and bcl-2 (17). Oxidative stress or redox dysregulation has beenreported to participate in apoptosis induction in several virus infections including HIV type I (18, 19) and HTLV-I (1, 20). Recently,treatment of embryonic stem cells with atRA has been reported toincrease intracellular ROIs (21). Furthermore, HTLV-I-positive cellsare even more susceptible to oxidative stress (20, 22). This evidenceprompted us to test the hypothesis that oxidative stress may beinvolved in l3cRA-driven growth inhibition and/or cell death of

HTLV-I-positive lymphocytes. Here we report that l3cRA is a potentapoptosis inducer for HTLV-I-positive cells and have investigatedwhether intracellular redox status may play a role in l3cRA-inducedapoptosis.

MATERIALS AND METHODS

Cells. PBMCswere isolatedfromperipheralvenous blood of fourpatientswith acute AU as well as HTLV-I-negative healthy volunteers by Ficoll

Hypaque density gradient centrifugation. PBMCs, ATL2, a HTLV-I-positiveT-cell line that was kindly provided by M. Maeda (Kyoto University, Kyoto,Japan), and Jurkat, a HTLV-I-negativeT-cell line, were cultured in RPM! 1640(Life Technologies, Inc., Grand Island, NY) supplemented with 10% heatinactivated FCS (Filtron, Brooklin, Australia), 100 units/ml penicillin G, and

0.1 @xg/mlstreptomycin at 37°Cin a humidified atmosphere of 5% CO2.Reagents. 13cRA and atRA, purchased from Sigma Chemical Co. (St.

Louis, MO), were dissolved at l0_2 M in 100% DMSO. The final DMSOconcentrations in the medium were no higher than 0. 1% in which control

experiments showed no effect on the proliferation or viability of the cells used(data not shown). BSO, NAC, and catalase (bovine liver, thymol-free; 0.048@xg/unit) were also purchased from Sigma Chemical Co. and dissolved in PBS.

PHA and rIL-2 were purchased from Life Technologies, Inc. and Genzyme(Cambridge, MA), respectively.

Proliferation Assay and Evaluation of the Percentage of Viable Cells.

The proliferation of PBMCs was evaluated by [3H]thymidine incorporation asdescribed. Briefly, 100 ,xl of 106cells/mI were cultured in 96-well microplates(Nunc, Naperville, IL) in triplicate samples for the number of indicated hours,pulsed with [3H]thymidine at 1 pCi/well for 4 h, and harvested. Incorporated[3H]thymidine was counted in a liquid scintillation counter (Aloka, Tokyo,

Japan). Viable cell numbers and the percentage of viable cells were assessedby trypan blue dye exclusion test using a hemacytometer.

Quantitation of the Percentage of Apoptotic Cells. The percentage ofapoptotic cells was assessed by fluorescence microscopy, after staining withAO and EB, as described previously (23). Briefly, 1 pJ of stock solutioncontaining 100 @xg/mlAO and 100 @xg/mlEB was added to 25 @xlof cellsuspension. After mixing, the cells were examined by fluorescence microscopy

(Nikon, Garden City, NY). Total cells as well as apoptotic cells that showedthe characteristic appearance of apoptosis, including cell shrinkage, blebbing,and apoptotic bodies, were counted manually.

4916

Role of Intracellular Redox Status in Apoptosis Induction of Human T-CellLeukemia Virus Type I-infected Lymphocytes by 13-cis-Retinoic Acid1

Keizo Furuke,2 Tetsuro Sasada, Yasuyo Ueda-Taniguchi, Akira Yamauchi, Takashi Inamoto, Yoshio Yamaoka,Hiroshi Masutani, and Junji Yodoi3

Department of Biological Responses. Institute for Virus Research (K. F., 1'. S., H. M., J. Y.), Department of Gastroenterological Surgery, Graduate School of Medicine (K. F..A. Y.. Y. Y.J, and College of Medical Technology, Kyoto University. Shogoin, Sakyoku, Kyoto 606-01 (T. I.], and the 2nd Department of internal Medicine, School of Medicine,Iwate Medical University, 19.1 Uchimaru, Morioka 020, (1'. U-Ti. Japan

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OXIDATIVE STRESS IN RA-INDUCED APOPTOSIS

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Fig. 1. The effect of l3cRA on proliferation and cell viability of PBMCs from four different patients with All (a) and PHA- or IL-2-activated PBMCs from a HTLV-I-negativehealthy donor (b). PBMCs (lOs cells/nil) from patients with ATh were cultured with or without the indicated concentrations of 13cRA for 4 days and then harvested for l3Hlthymidineincorporation and trypan blue dye exclusion. PBMCs (106 cells/ml) from healthy donors were cultured with or without the indicated concentrations of I3cRA in the presence of 0.1%PHA or 100 lU/mi rlL-2 for 3 days and then harvested for [3Hlthymidine incorporation and trypan blue dye exclusion. A representative of four different healthy donors that were allcomparable is shown. Columns, [H3]thymidine incorporation; 0, percentage of viable cells.

The proportion of cells undergoing apoptosis was also measured by flowcytometric analysis after staining with P1 (Sigma) as described previously (24).In brief, cell pellets were suspended in 1 ml of hypotonic fluorochromesolution containing 50 @.sg/miP1in 3.4 mistsodium citrate, 0.1% Triton X-l00,1 mM Tris (pH 8), and 0.1 mrsi EDTA and incubated in the dark at 4°Covernight. The P1 fluorescence of individual nuclei was measured, and the

percentage of cells with hypodiploid DNA was calculated with a flow cytometer (Ortho, Raritan, NJ).

Immunoblot Analysis. Immunoblotanalysis was performedas describedpreviously (25). Briefly, 106cells were lysed on ice in 0.5% NP4Olysis buffercontaining 50 mM Tris-HCI (pH 7.5), 150 mM NaCI, I mxi EGTA, 50 @.tg/mlleupeptin, 50 @g/mlaprotinin, and 50 msi p-nitrophenyl-p'-guanidinobenzo

4917

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OXIDATIVE STRESS IN RA-INDUCED APOFTOSIS

with 70% ethanol, air-dried, and dissolved in 15 @ilof 10 mM Tris-HCI with 1

mM EDTA (pH 7.5). After digesting RNA with RNase A (0.6 mg/ml at 37°Cfor 30 mm), the sample was electrophoresed in a 2% agarose gel with TAE

buffer [50 mM Tris-HCI, 20 nmi sodium acetate, 2 mrsiEDTA, and 18 misi NaCl(pH 8.05)1.DNA was stained with EB and visualized by UV light.

Measurement of Intracellular GSH. Intracellular GSH contents weremeasured using the Glutathione Assay kit (Calbiochem, San Diego, CA). In

brief, 2 X 106 cells treated with the indicated doses of RAs, BSO, and/or NACfor 48 h were homogenized in 5% metaphosphoric acid using a Teflon pestle.Cell lysates were assayed according to the manufacturer's instructions.

Detection of Peroxides Accumulated in ATL2 Cells by Flow Cytometry.ATL2 cells (2 X l0@ cells/mI) were cultured with or without indicatedtreatments for 24 h and washed with PBS. Then cells were recultured in fresh

RPM! 1640with 10%FCS and 5 ,.LMDCFH-diacetate (Molecular Probes, Inc.,Eugene, OR) for 30 mm at 37°C,and the fluorescence intensity was measuredby flow cytometer.

RESULTS

13cRA Inhibited Proliferation and Induced Cell Death ofPBMCs from Patients with Acute ATL but not PBMCs fromNormal Donors. We have shown that 13cRA inhibited proliferationof HTLV-I-positive but not HTLV-I-negative T-cell lines (2). Todetermine whether l3cRA-induced growth inhibition is generalizableto other HTLV-I-infected lymphocytes, we investigated the effects ofl3cRA on cell growth and viability of PBMCs obtained from fourpatients with acute All. Although there was individual variability

4918

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Fig. 2. The effects of l3cRA and atRA on cell proliferation of ATL2 (a), a HTLV-Ipositive cell line. and Jurkat (b), a HTLV-I-negative cell line. ATL2 cells (2 X l0@cells/ml) and Jurkat cells (I X 10' cells/ml) were cultured and counted serially by trypanblue dye exclusion for 6 days. The means and SD (vertical bars) among triplicate samplesin a representative of four different experiments that were all comparable are shown. c;@ 10-8i@'@10.6iø-@

ate, and equivalent amounts of total cellular protein (50 @.sg)were separated on12% SDS-polyacrylamide gels, blotted to polyvinyl difluoride membranes(Millipore, Bedford, MA), and incubated with antihuman Bcl-2 monoclonal

Ab (Calbiochem, Cambridge, MA) or rabbit antihuman Bax polyclonal Ab(Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were incubatedwith appropriate horseradish peroxide-conjugated second Abs. followed by

reaction with enhanced chemiluminescence detection reagents (Amersham,Buckinghamshire, United Kingdom) and exposure to XAR film (Eastman

KOdak, Rochester, NY) for 30 s.

Flow Cytometry Analysis. For measuringFas expression, 106cells werestained with FITC-conjugated antihuman Fas monoclonal Ab (Immunotech,

Westbrook, ME) in PBS with 1% FCS and 0.1% sodium azide on ice for 20

mm.Thefluorescenceintensitywasmeasuredbyflowcytometer(BectonDickinson, San Jose, CA).

DNA Fragmentation Assay. DNA fragmentationwas detectedby agarosegel electrophoresis as described previously (26). In brief, 5 X 106cells wereharvested, centrifuged, and washed with cold PBS. The cell pellet was lysed

with a buffer consisting of 10 nmi Tris-HCI, 10 mMEDTA, and 0.2% Tritonx-100 (pH 7.5) and centrifugedat 13,000X g at 4°C.Next,the supernatantwas extracted, first with phenol, and then with phenol-chloroform:isoamylalcohol (24:1). The aqueous phase was brought to 300 mM NaC1 and precip

itated with 2 volumes of ethanol overnight at —20°C.The pellet was rinsed

hour

a)

2 —. 13cRA

j: -“O―--atRA

b)

.@ ....o.... noRA

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@ —..-—— l3cRA

Fig. 3. The dose response and time course of RA-induced apoptosis of ATL2 cells. a,2 X l0@cells/nil ATL2 cells were cultured in the presence or absence of the indicatedconcentration of l3cRA or atRA. After 48 h of culture, cells were harvested for P1staining. b, 2 X l0@cells/mI ATL2 cells were cultured in the presence or absence of l0@M 13cRA. Cells were harvested at the indicated time points for P1 staining.

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OXIDATIVE STRESS IN RA-INDUCED APOPTOSIS

among the patients, l3cRA inhibited the proliferation and reduced thepercentage of viable cells of PBMCs in a dose-dependent manner inall four cases (Fig. la). In contrast, 13cRA did not alter the viabilityof unstimulated PBMCs obtained from HTLV-I-negative healthy donors (data not shown). In addition, to evaluate whether l3cRA affectsthe physiological response of PBMCs to mitogens or IL-2, we furthertested the effects of 13cRA on proliferation and viability of PHA- orrIL-2-activated PBMCs from HTLV-I-negative donors. As shown in

Fig. lb. 13cRA did not significantly affect their proliferation orviability, suggesting that 13cRA can selectively induce apoptosis ofHTLV-I-infected cells without suppression of normal immuneresponse.

13cRA Induced Apoptosis of ATL2 Cells More Potently thanDid atRA. To elucidate the mechanism by which l3cRA induces thedeath of HTLV-I-positive lymphocytes, we used ATL2 cells as amodel. We first confirmed that 13cRA inhibited the proliferation ofATL2 cells but not of HTLV-I-negative Jurkat cells (Fig. 2, a and b).Next we showed that l3cRA induced apoptosis of ATL2 cells in 48 hin a dose-dependent manner (Fig. 3, a and b). As shown in Fig. 3a,

13cRA had more potent activity in apoptosis induction than did atRA,

which is known to exert a variety of biological effects through bindingto nuclear receptor RAR. Because the binding affinity of l3cRA toRAR is much lower than that of atRA (1 1), an explanation of thisresult may be related to their solubility, cell uptake of ligands, metabolism of ligands, RAR isoform selectivity of 13cRA versus atRA,or a pathway distinct from RAR.

RAs Did Not Affect the Expression of Bcl-2, Bax, or Fas inATL2 Cells. A number of pathways leading to apoptosis are regulated by the expression of Bcl-2 as well as Bax (27, 28). Therefore, weexamined the effect of 13cRA or atRA on the expression of Bcl-2 or

Bax in ATL2 cells by immunoblot analysis. As shown in Fig. 4a,neither RA affected the expression of either Bcl-2 or Bax at 10 p.M. atwhich they induced apoptosis of ATL2 cells. We also investigated theexpression of Fas on the surface of ATL2 cells treated with RAs byflow cytometry, because Fas plays a major role in the induction ofapoptosis, particularly in activated lymphocytes (29). However, RAsdid not show any effects on their Fas expression (Fig. 4b).

BSO Enhanced 13cRA-induced Apoptosis of ATL2 Cells. Thereis growing evidence that the intracellular redox status plays an im

portant role in the regulation of cell death ( I5, 30, 3 1). GSH is one ofthe major components controlling intracellular redox status (32).Therefore, we next examined the effects of NAC and BSO, which areknown to increase and decrease intracellular GSH contents, respectively (33, 34), on l3cR.A-induced apoptosis. Fluorescence microscopy (Fig. 5a) as well as flow cytometry (Fig. 5b) revealed that acombination of 13cRA with 500 @.LMBSO resulted in a higher percentage of apoptosis than did l3cRA alone. Furthermore, whereasDNA ladder formation was not detected after a single treatment withl0@ M l3cRA, it was clearly detectable in cells treated with the sameamount of l3cRA in the presence of 500 @.tMBSO (Fig. 5c). BSOalone had no ability or only a weak ability to induce apoptosis (Fig.5, a—c).Furthermore, in the presence of 10 mr@iof NAC, the percentage of apoptosis of 13cRA-treated ATL2 cells was decreased by nomore than about 20% (Fig. 5a). These findings suggest that intracel

lular GSH may modulate apoptosis induction by l3cRA, althoughGSH imbalance alone does not lead to apoptosis. Augmentation ofapoptosis by BSO was also seen in the case of atRA, although thepercentage of apoptosis was lower than that of 13cRA (data notshown), suggesting that apoptosis induction by atRA may involve amechanism similar to l3cRA.

Intracellular GSH Content Was Not Altered in 13cRA-treatedATL2 Cells but Was Decreased by BSO. To confirm whetherintracellular GSH is involved in 13cRA-induced apoptosis, we meas

4919

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Fig. 4. a, the expression of either Bcl-2 or Bax in RA-treated ATL2 cells. ATL2 cells(106) were lysed after treatment with or without 10 @iMl3cRA or atRA for 24 h andsubjected to immunoblot analysis as described in “Materialsand Methods.―b, theexpression of Fas in RA-treated ATL2 cells. ATL2 cells (106) were cultured with orwithout 10 jiM I3cRA or atRA for 24 h and harvested for staining with FITC-conjugatedanti-Faa Ab. Shaded histograms show unstained cells as a background.

I3cRA

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0 0.01 0.1 1 10

Table I Measurement of intracellular GSH contents in treated ATL2 cellsATL2 cells were cultured with the indicated concentrations of 13cRA in the presence

or absence of BSO or NAC for 48 h. Intracellular GSH contents (nmol/l06 cells) weremeasured as described in “Materialsand Methods.―The means ±SD of three independentexperiments areshown.l3cRA

(jiM) Control― 500 ,sMBSO 10 instNAC0

4.37 ±0.45 1.90 ±0.21 3.85 ±0.350.01 3.96 ±0.38 2.15 ±0.25 4.20 ±0.450.1 3.96 ±0.32 2.06 ±0.20 3.95 ±0.381 4.15 ±0.35 1.97 ±0.18 4.05 ±0.3710 3.91 ±0.45 2.00 ±0.23 3.93 ±0.41a

ATL2 cells were treatedwith the indicatedconcentrationsof l3cRA alone.

OXIDATIVE STRESS IN RA.INDUCED APOFTOSIS

a

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Fig. 5. a, the effects of BSO or NAC on the percentage of apoptosis of 13cRA-treatedATL2 cells. ATL2 cells (2 X 10―cells/mI) were cultured in the presence or absence of 500@LMBSO or 10 mM NAC and harvested for AO and EB staining after 48 h ofculture. The means and SD (verticalbars) among three different experiments are shown. b, flow cytometric

analysis of l3cRA-induced apoptosis of ATL2 cells. ATL2 cells were cultured with or without l0@ si 13cRA, 500 jiM BSO, and l0@ M l3cRA + 500 p.MBSO for 48 h and analyzedfor the percentage of hypodiploid DNA by flow cytometry after P1staining. c, DNA ladder formation in 13cRA- and/or BSO-treated ATL2 cells. DNA was isolated from ATL2 cellswith indicated treatments for 48 h and electrophoresed as described in “Materialsand Methods.―Lane I, molecular size marker; Lane 2, no treatment; Lane 3, 500 @MBSO; Lane 4,l0° M l3cRA; Lane 5. l0@ M l3cRA; Lane 6, lO_6 M 13cRA; Lane 7, l0@ M l3cRA + 500 @.u,iBSO.

ured intracellular GSH contents in untreated or treated ATL2 cells(Table 1). Whereas l0_8@l0_5 M l3cRA had no effects on intracellular GSH content, 500 p.M BSO alone or together with l3cRAdecreased it by approximately 50%, suggesting that BSO may facilitate l3cRA-induced apoptosis through the reduction of intracellular

GSH level. Next we examined the effect of NAC on intracellular GSHcontent. Even at 10 mat, NAC failed to alter GSH levels in ATL2cells. A limited inhibitory effect of NAC against I3cRA-inducedapoptosis (no more than 20% inhibition) may be explained by areducing activity of NAC itself rather than an increase in GSH. Theseresults suggest that whereas l3cRA alone does not significantly affectintracellular GSH contents, BSO may increase l3cRA-induced apoptosis by decreasing the intracellular GSH levels.

13cRA Alone Increased Intracellular Peroxide Accumulation,and BSO Enhanced It Further. We next assayed intracellular peroxides to assess whether l3cRA, in the absence or presence of BSO,modulates intracellular redox status in ATL2 cells through attenuationof peroxide levels. We first confirmed that the interference of DCFHfluorescence by RAs was not observed after pretreatment of ATL2cells with 1 and 10 p.M RAs for only 1 h and that a micromolarconcentration of retinol, which is another lipid-like agent similar to

4920

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1 2 3 4 5 6 7

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OXIDATIVE STRESS IN RA-INDUCED APOPTOSIS

added to the culture medium 1 h before treatment with l3cRA. Asshown in Fig. 7, pretreatment with catalase inhibited apoptosis induced by 13cRA in a dose-dependent manner (about 50% inhibitionby 10,000 units/mI catalase). This finding suggests that intracellularaccumulation of peroxides may be involved in cell death elicited byl3cRA.

DISCUSSION

p@@@ r@ 2 â€3̃@@ This study demonstrated the ability of l3cRA to induce cell death

)@ 10 10 10 10 of HTLV-I-positive cells, including PBMCs from patients with ATL

as well as the ATL2 cell line. In addition, 13cRA induced apoptosisofATL2cellsmorepotentlythandidatRA,despiteaweakbindingaffinity of 13cRA for the nuclear receptors (RAR or RXR). Moststudies of apoptosis induction by retinoids, such as atRA, 9cRA, or asynthetic RA, 4-hydroxyphenyl retinamide, have revealed that RARand/or RXR are involved in the signaling pathway leading to apop

tosis (35—39).Furthermore, recently, the activation of RAR and/orRXR has been shown to decrease the expression of Bcl-2 (40—42).Okazawa et a!. (43) reported that overexpression of Bcl-2 inhibitedatRA-induced apoptosis of embryonal stem cells. Thus, the downmodulation of Bcl-2 expression is believed to be one of the mechanisms of RA-induced apoptosis. However, as for 13cRA, there hasnever been evidence regarding the involvement of either the nuclearreceptors or Bcl-2 expression in the induction of apoptosis. Accordingto our experiments, at least protein levels of bcl-2 were not altered bytreatment with 13cRA or atRA, although it was still unclear whetherbcl-2 functions remained the same after the treatment. We have also

confirmed no effects of either 13cRA or atRA on the expression ofother apoptosis-related proteins, Bax and Fas. Because HTLV-Iinfected cells are known to be more susceptible to oxidative stressthan HTLV-I negative cells (1, 22), we explored the role of intracellular redox status in the induction of apoptosis by 13cRA.

Here we showed that BSO enhanced l3cRA-elicited apoptosis ofATL2 cells through the reduction of intracellular GSH levels, whereas13cRA alone did not affect GSH contents. These results suggest thatintracellular GSH levels may modulate the sensitivity of ATL2 tol3cRA. GSH, a free radical scavenger, is required for protecting cellsfrom oxidative stress (32). BSO has been reported to enhance theanticancer effects of several drugs such as Adriamycin, melphalan,

RAs, did not show any effects on apoptosis induction or peroxidegeneration (data not shown). As shown in Fig. 6, l3cRA as well asBSO increased intracellular peroxide accumulation in the 24 h afterthe treatment. The ability of 13cRA to increase peroxide contents wasmore potent than that of atRA. BSO also had an additive effect on theaccumulation of peroxides in l3cRA-treated cells. These results indicate that the ability of l3cRA or atRA to induce apoptosis of ATL2

cells correlated with the increase of intracellular peroxide contents.Therefore, we hypothesized that 13cRA-induced production of peroxides might result in apoptosis induction in ATL2 cells.

Pretreatment with Catalase PrOtected 13cRA-treated ATL2Cells from Apoptosis. To confirm the hypothesis, we examinedwhether catalase, which quenches hydrogen peroxide, blocks 13cRAinduced apoptosis of ATL2 cells. Catalase (500—10,000units/mi) was

0

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Fig. 6. Measurement of intracellular peroxide accumulation by flow cytometry. ATL2cells (2 X l0@cells/nd) were cultured with or without the indicated reagents for 24 h. andDCFH-diacetate (5 jiM) was added to the medium, followed by incubation for 30 mm at37°C.Intracellular peroxides were detected by flow cytometer. A representative of threeindependent experiments that were all comparable is shown. The result of untreated ATL2cells is shown by an unshaded histogram in each graph.

Catalase (U/mI)

Fig. 7. The effect of extracellular catalase on 13cRA-induced apoptosis. After ATL2cells (2 X lO@cells/mI) were cultured in the presence or absence of the indicatedconcentrations of catalase for I h, l06 M I3cRA was added for an additional 48 and 60 h.Then cells were harvested for AO and EB stainingto detemsinethe percentageof apoptosis.The means and SD (vertical bars) among three differentexperimentsare shown.

4921

1 Lt\I atRA

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OXIDATIVE STRESS IN RA.INDUCED APOPTOSIS

and cisplatin, most of which are known to induce apoptosis by theproduction of ROIs (44—46). Therefore, to elucidate whether ROl

production is involved in I3cRA-induced apoptosis, we measuredintracellular peroxide levels in l3cRA-treated ATL2 cells. Interestingly, flow cytometric analysis revealed that l3cRA treatment induced the accumulation of intracellular peroxide products in 24 h

more potently than did atRA and that BSO enhanced them further.The increase in peroxides preceded the induction of apoptosis that wasdetected 48 h after treatment. In addition, pretreatment with catalase,which has been reported to affect the intracellular redox status (16,47), partially protected l3cRA-treated ATL2 cells from apoptosis.These results suggest that generation of peroxides or oxidative stress

is an important biological step in 13cRA-induced apoptosis of HTLV

I-positive lymphocytes. Furthermore, our data are consistent with

other reports regarding the relationship of oxidative stress to RAinduced apoptosis. Delia et a!. showed that horseradish peroxidaseinduced apoptosis was inhibited by some antioxidants, including

NAC, ascorbic acid, a-tocopherol, and deferoxamine (40). The apoptosis of embryonic stem cells by atRA was reported to be blocked bycatalase, superoxide dismutase, or phenol (2 1). Therefore, ROI generation, i.e. , oxidative stress, may generally occur by treatment withRAs.

We have reported that l3cRA potently inhibits the TRXfl'RX reductase (TxR) system (2, 48), which protects cells against oxidative stressthrough scavenging ROIs (12, 13, 49, 50), in cell-free studies. Therefore,it is possible that suppression of the activity ofTRXTFxR may participate

in the increase in peroxides by l3cRA. By contrast, we found that l3cRA

did not decrease the intracellular GSH levels, despite a rise in intracellular 18.

peroxides, suggesting that the GSH/GSH reductase system may not beaffected in the presence of l3cRA. Therefore, the results that BSOenhanced both apoptosis induction and peroxide generation by l3cRAcan be explained by dysregulation of these two critical redox systems.

However, l3cRA may encourage peroxide production via another mechanism like respiratory burst, because l3cRA and atRA were reported toincrease phagocytosis and production of ROIs in human endothelial cells

(5 1). We need more investigations as to how oxidative stress is generatedafter RA treatment.

In conclusion, we propose that l3cRA leads to apoptosis of HTLVI-infected lymphocytes through the generation of peroxides, i.e., oxidative stress. It is suggested that the intracellular GSH contents may

modulate l3cRA-induced apoptosis. Combined treatment with l3cRAand BSO is a potent inducer of apoptosis in HTLV-I-infectedlymphocytes and may be a potential therapy for HTLV-I-related

disorders.

ACKNOWLEDGMENTS

We are very grateful to Drs. M. Matsuoka (Kumamoto University,Kumamoto, Japan) and K. Takatsuki (Kitano Hospital, Osaka, Japan) for

providing peripheral venous blood from patients with ATh. We thank Dr. EdaT. Bloom (Center for Biologics Evaluation and Research, Food and DrugAdministration) for critical review of the manuscript.

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1997;57:4916-4923. Cancer Res Keizo Furuke, Tetsuro Sasada, Yasuyo Ueda-Taniguchi, et al.

-Retinoic Acidcis13-Human T-Cell Leukemia Virus Type I-infected Lymphocytes by Role of Intracellular Redox Status in Apoptosis Induction of

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