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Biochemical and Biophysical Research Communications 286, 726–734 (2001)

doi:10.1006/bbrc.2001.5416, available online at http://www.idealibrary.com on

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rsenic Trioxide Induces G2/M Growth Arrest andpoptosis after Caspase-3 Activation and Bcl-2hosphorylation in Promonocytic U937 Cells

ong-Wook Park,* Yun-Jung Choi,* Min Ah Jang,* Suk-Hwan Baek,† Jun Hee Lim,†ony Passaniti,‡ and Taeg Kyu Kwon*,1

Department of Immunology, School of Medicine, Keimyung University, Taegu, South Korea; †Department of Biochemistrynd Molecular Biology, College of Medicine, Yeungnam University, Taegu, South Korea; and ‡University of Marylandchool of Medicine, Greenebaum Cancer Center, Baltimore, Maryland 21201

eceived July 5, 2001

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Arsenic trioxide has recently been shown to inhibitrowth and induce apoptosis in acute promyelocyticeukemia (APL), but little is known about the molecu-ar mechanisms mediating these effects. Here we dem-nstrate that treatment of promonocytic U937 cellsith arsenic trioxide leads to G2/M arrest which wasssociated with a dramatic increase in the levels ofyclin B and cyclin B-dependent kinase and apoptosis.e further show that apoptosis occurs after bcl-2

hosphorylation and caspase-3 activation followed byleavage of PARP and PLC-g1 degradation and DNAragmentation. The arsenic trioxide-induced apopto-is could be blocked by the protein synthesis inhibitorycloheximide. In addition, pretreatment of U937 cellsith the DNA polymerase inhibitor aphidicolin alsolocked apoptosis, but did not cause the arrest of cellsn the G2/M phase. The findings suggest that arsenicrioxide exerts its growth-inhibitory effects by modu-ating expression and/or activity of several key G2/Megulatory proteins. Furthermore, arsenic trioxide-ediated G2/M arrest correlates with the onset of

poptosis. © 2001 Academic Press

Key Words: arsenic trioxide; apoptosis; bcl-2 phos-horylation; cell cycle; leukemia.

Recently, low concentration (micromolar or less) ofs2O3 have been reported to induce complete remission

n a high proportion of patients with acute promyelo-ytic leukemia (APL) without severe toxicity (1–4).PL constitutes 10 to 15 percent of the cases of acuteyeloid leukemia in adults. APL is associated with a

pecific t(15;17) translocation which results in a fusion

1 To whom correspondence should be addressed at Departmentf Immunology, School of Medicine, Keimyung University, 194ongSan-Dong Jung-Gu, Taegu, 700-712, South Korea. Fax: 82-53-55-1398. E-mail: [email protected].

726006-291X/01 $35.00opyright © 2001 by Academic Pressll rights of reproduction in any form reserved.

f the PML (promyelocytic leukemia) gene and retinoiccid receptor a gene to yield a PML/RARa fusion pro-ein (5, 6). This fusion protein causes arrest of matu-ation at the promyelocyte stage of myeloid develop-ent (7, 8).Arsenic trioxide has been shown to be clinically ef-

ective in APL treatment by inducing of apoptosis (3).owever, it remained to be elucidated how arsenic

rioxide caused apoptosis. One mechanism to be con-idered was that arsenic trioxide could induce apopto-is via the cellular glutathione redox system (9, 10).he ability of arsenic trioxide to induce apoptosis in

eukemic cells is dependent on the activity of the en-ymes that regulate cellular H2O2 content (9, 10). Ar-enic trioxide induced apoptosis in B-cell leukemia cellines in vitro in a manner that involved activation ofaspases and down-regulation of bcl-2 protein expres-ion (11, 12).Progression through the mammalian cell cycle is

acilitated by cyclin/cyclin dependent kinase (cdk) com-lexes each of which activated at a specific point duringhe cell cycle (13, 14). The kinase activity of cdc/cyclin

is activated specially at the G2/M transition (13, 14).icrotubule inhibitor, such as taxol and nocodazole,

timulate p34cdc2 kinase activity and arrest the cellycle at G2/M phase (15, 16). Recently, Li et al. re-orted that arsenic trioxide may act as a noncompeti-ive inhibitor of GTP binding to tubulin. Additionally,t was found that the arsenic trioxide mediated mitoticrrest could be prevented by addition of an exogenousTP (17).Here we report that arsenic trioxide arrests cell di-

ision at the G2/M phase and that it induces apoptosisia caspase-3 activation and bcl-2 phosphorylation inromonocytic U937 cells. The arsenic trioxide medi-ted G2/M phase arrest of U937 cells was linked to thectivation of cyclin B-dependent kinase and accumula-

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Vol. 286, No. 4, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ion of cyclin B. Our data show that arsenic trioxideauses activation of cyclin B-dependent kinase andccumulation of cyclin B in association with mitoticrrest. The arsenic trioxide mediated G2/M phase ar-est also correlates with the onset of apoptosis.

ATERIALS AND METHODS

Cell culture, cell treatments, and materials. The human leukemia937 cells were obtained from ATCC (Rockville, MD). The cultureedium used throughout these experiments was Dulbecco’s modifiedagle medium containing 10% FCS, 20 mM HEPES, 100 mg/mlentamicin (complete medium). Anti-cdk2, anti-cdc2, anti-cyclin A,nti-cyclin B, anti-bcl-2, and anti-PARP antibodies were purchasedrom Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-bcl-xLnd anti-caspase-3 were purchased from Oncogene Science (Cam-ridge, MA). Lysine-rich histone was purchased from United Statesiochemical Corporation (Cleveland, OH) and protein G-agarose

rom Calbiochem (La Jolla, CA). Arsenic trioxide, aphidicolin andycloheximide were purchased from Sigma (St. Louis, MO).

Immunoprecipitation and Western blotting. Cellular lysates wererepared by suspending 1 3 106 cells in 100 ml of lysis buffer (137M NaCl, 15 mM EGTA, 0.1 mM sodium orthovanadate, 15 mMgCl2, 0.1% Triton X-100, 25 mM Mops, 100 mM phenylmethylsul-

onyl fluoride, and 20 mM leupeptin, adjusted to pH 7.2). The cellsere disrupted by sonication and extracted at 4°C for 30 min. For

mmunoprecipitation, 100 mg of cellular lysate was allowed to reactith 2 mg of antibody at 4°C for 1 h. The immune complexes were

ecovered by the addition of protein G-agarose beads. The complexeads were washed, suspended in Laemmli buffer, boiled for 5 minnd the proteins were separated in 10% SDS–polyacrylamide gels.he proteins were electrotransferred to Immobilon-P membranes

Millipore Corp., Bedford, MA). Detection of specific proteins wasarried out with an ECL Western blotting kit according to manufac-urer’s instructions.

Kinase assay. The immunoprecipitates obtained from 100 mg ofell lysate as described above were assayed for kinase activity (18).riefly, immunecomplexes were incubated in HB buffer (60 mM-glycerophosphate, 15 mM p-nitrophenylphosphate, 15 mM MgCl2,.1 mM sodium orthovanadate, 80 mM NaCl, 100 mM phenylmeth-lsulfonyl fluoride, and 20 mM leupeptin, 5 mM EGTA, 25 mM Mops,H 7.2), 50 mM ATP, 5 mCi [g-32P] ATP (10 Ci/mM: NEN DuPont,ilmington, DE) containing histone H1 protein at 30°C for 30 min.

he reactions were terminated by the addition of 15 ml of SDS–PAGEample buffer. Proteins were separated by 10% SDS–PAGE, andhosphorylations was detected by autoradiography after exposure tolm at 270°C.

Flow cytometry analysis and DNA fragmentation. Approximately3 106 U937 cells were suspended in 100 ml of PBS, and 200 ml of

5% ethanol were added while vortexing. The cells were incubated at°C for 1 h, washed with PBS, and resuspended in 250 ml of 1.12%odium citrate buffer (pH 8.4) together with 12.5 mg of RNase.ncubation was continued at 37°C for 30 min. The cellular DNA washen stained by applying 250 ml of propidium iodide (50 mg/ml) for 30in at room temperature. The stained cells were analyzed on aACScan flow cytometer for relative DNA content based on in-reased red fluorescence. Assessment of apoptosis was determined byonitoring the sub-diploid population by flow cytometer. Cell cycle

nalysis was performed by flow cytometer using a commerciallyvailable software package (Modfit; Verity Winlist, Topsham, ME).ragmented cytoplasmic DNA was prepared essentially as described

19) and analyzed electrophoretically on 2% agarose gels containing.1 mg/ml of ethidium bromide.

Caspase-3 activity assay. To evaluate caspase-3 activity, cell ly-ates were prepared after their respective treatment with As2O3.

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g of cell lysates in 100 ml of reaction buffer (1% NP-40, 20 mMris–HCl, pH 7.5, 137 mM NaCl, 10% glycerol) containing theaspase-3 substrate (DEVD-pNA) at 5 mM. Lysates were incubatedt 37°C for 2 h. Thereafter, the absorbance at 405 nm was measuredith a spectrophotometer.

ESULTS

2/M Phase Arrest and Apoptosis Are Inducedby Arsenic Trioxide

To set up the experimental conditions for investigat-ng the relationship between G2/M phase arrest andpoptosis in U937 cells, we carried out dose-responsetudies of the G2/M arrest and apoptosis effect of ar-enic trioxide on U937 cells. Cells were incubated inhe absence or presence of arsenic trioxide (0.5–6 mM)t 37°C for 24 h. As the dose of As2O3 increased from.5 mM to 6 mM, there was a parallel increase in G2/Mhase population. As shown in Fig. 1A, 50% of controlells were in G1 phase, 40% were in S phase, and 10%ere in G2/M phase. The cells treated with 4 mM As2O3

howed an accumulation of cells in the G2/M phase ofhe cell cycle such that by 24 h, 58% of the cells were in2/M phase. Transition through the G2/M phase of the

ell cycle has recently been shown to involve cdc2 ki-ase. Cyclin A and cyclin B are the positive regulatorsf cdc2 kinase, and they function in both the S and2/M phase transition (13, 14). Because As2O3 treat-ent results in G2/M phase arrest, we investigated the

ssociation of drug-induced G2/M phase arrest withlternations in cyclin expression and function. Ashown in Fig. 1B, As2O3 treatment resulted in a mark-dly increased accumulation of cyclin B protein, whichccurred in a concentration-dependent manner. Treat-ent with As2O3 did not increase the amount of de-

ectable cdc2, cdk2 and cyclin A. Subsequently, weetermined cdc2 kinase activity, as described underaterials and Methods. As shown in Fig. 1C, control

ells displayed low cdc2 kinase activity. However, theinase activity was increased significantly in cellsreated with 4 mM As2O3. These results indicate thathe G2/M phase arrest caused by As2O3 is associatedith increased accumulation of cyclin B and stimula-

ion of cdc2 kinase activity.To visualization of apoptotic cells, U937 cells were

xposed to various concentrations of As2O3 for 24 h.s2O3-induced apoptosis was indicated by DNA distri-ution as revealed by FACS analysis demonstratingypodiploid DNA and DNA fragmentation. The bio-hemical hallmark for apoptosis is the cleavage of DNAt an internucleosomal ladder. We therefore deter-ined whether As2O3 would induce such DNA frag-entation. As shown in Fig. 2A, 1 mM As2O3 had no

ffect on cell apoptosis, whereas 2 mM As2O3 moder-tely increased the percentage of apoptotic cells andevealed fragmented DNA. Various proteins are in-

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olved in the execution of apoptosis. Two of the mostxtensively studied are bcl-2 and caspase-3. They playn important role in the apoptotic response of U937ells to a variety of stimuli and severs as an earlyarkers of the induction of apoptosis (20, 21). To iden-

ify what would be a possible mechanism underlyinghe As2O3 induction of apoptosis, we performed a West-rn blot analysis of bcl-XL, bcl-2 and caspase-3 expres-ion in U937 cells treated with various concentrationsf As2O3. The various concentrations of As2O3 used didot reduce the levels of bcl-XL and bcl-2 protein in theells, but 4 and 6 mM As2O3 induce phosphorylation ofcl-2. Phosphorylation of bcl-2 reduces its ability toorm heterodimers with bax (22–24). Caspase-3 is ac-ivated by proteolytic processing of the 32 kDa formnto small two subunits. Activity of caspase-3 duringrsenic trioxide induced apoptosis was examined as

decrease in proenzyme level using Western blotnalysis. Treatment with As2O3 did not reduced themount of detectable proenzyme levels of caspase-3,ut it did cause a slight decrease in the levels ofaspase-3 in cells exposed to 4 or 6 mM As2O3 for 24 h.o further investigate and quantitate the proteolyticctivity of caspase-3, we performed an in vitro assayased on the proteolytic cleavage of DEVD-pN byaspase-3 into the chromophore p-nitroanilide (pNA).s shown in Fig. 2C, 2 mM As2O3 or 4 mM As2O3

reatment caused 3 and 4-fold increases in caspase-3ctivity, respectively. During apoptosis, poly(ADP-ibose) polymerase (PARP) and PLC-g1 are producedy cleavage by activated caspase-3 (25, 26). The cleav-ge of PARP and PLC-g1 was observed in cells exposed

FIG. 1. Arsenic trioxide induces G/M phase arrest. Flow cytometreated for 24 h with various concentrations of As2O3. The fraction ofhree independent experiments and bars represent standard deviatioells were treated with the indicated concentrations of As2O3. Equalnd analyzed by Western blot for cdk2, cdc2, cyclin A, and cyclin B. (Cells were immunoprecipitated with cdc2 and assayed for kinase act

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o 2 mM As2O3 for 24 h. These results suggest thatpoptosis induced by As2O3 is associated with bcl-2hosphorylation, caspase-3 activation, and PARPleavage.

inetics of the G2/M Arrest and Apoptosisin Arsenic Trioxide-Treated Cells

To investigate the relationship between G2/M phaserrest and apoptosis in U937 cells, we carried out timeinetic studies of the G2/M arrest and apoptosis effectsf arsenic trioxide on U937 cells. Exponentially grow-ng U937 cells were treated with 4 mM As2O3 andubjected to cell cycle analysis at 6, 12, 18, and 24 h byACS. Untreated cells showed a very low ratio of apop-osis, whereas the cells treated with 4 mM As2O3

howed a progressive increase in the apoptosis ratio.ultivation of the cells with 4 mM As2O3 increased theopulation of U937 cells in the G2/M phase in a timeependent manner, while it decreased the populationn the G1 phase (Fig. 3A).

We then looked for effects of As2O3 on the expressionf dominant regulatory proteins participating in G2/Mhase or apoptosis related events. Total proteins werextracted and analyzed by Western blot for cyclin B,cl-2, caspase-3 and PARP. As shown in Fig. 3B, thereas a low level of cyclin B detectable in untreated937 cells, which increased after treatment withs2O3 reaching maximal levels at 18 and 24 h. The

evels of cdc2 and cdk2 in U937 cells did not changepon As2O3 treatment. Bcl-2 phosphorylation was de-ected after 12 h of treatment. There was no detectable

analysis of the DNA content of untreated U937 cells (con) and cellsls in G2/M phase as indicated. Data are mean values obtained from(B) Effect of As2O3 on cell cycle regulatory protein expression levels.ounts of whole cell lysates (50 mg) were subjected to electrophoresisual amounts of cell lysates (100 mg) from As2O3-treated or untreatedy using histone H1 as substrate.

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hange in proenzyme level of caspase-3 until after 18 hf As2O3 treatment, and caspase-3 levels were slightlyecreased in cells treated for 24 h. However, cleavagef PARP was observed 12 h after the start of treatmentith 4 mM As2O3, although a caspase-3 cleavage wasot detected at that time point. It is possible that otheraspase-3 participates in the As2O3 induced cleavage ofARP.

aspase-3-Mediated Apoptosis Inducedby Arsenic Trioxide

To address the significance of caspase-3 activation ins2O3 induced apoptosis, we used a general and potent

nhibitor of caspases, z-VAD-fmk (benzyloxycarbony-al-Ala-Asp-fluoromethyl ketone). 4 mM As2O3 stronglytimulated caspase-3 protease activities, but z-VAD-mk pretreated cells abolished As2O3-induced caspase-3ctivities (Fig. 4A). Blockade of caspase-3 activities byretreatment of U937 cells with 50 mM z-VAD-fmkrevented 4 mM As2O3-induced cleavage of PLC-g1

FIG. 2. Arsenic trioxide induces apoptosis. Apoptosis and DNytometer. Data are mean values from three independent experimxtracted and analyzed on 2% agarose gel. (B) Effect of As2O3 on andicated concentrations of As2O3. Equal amounts of whole cell lysalot for bcl-xL, bcl-2, caspase-3, PARP, and PLC-g1. (C) U937 cells wn lysis buffer. Enzymatic activities of caspase-3 were determined byDEVD-pN) in a 100 ml assay buffer for 2 h at 37°C. The release of ch405 nm). Data are mean values from three independent experimen

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Fig. 4B). These data clearly indicate that As2O3-inducedpoptosis is associated with caspase-3 activation.

rotein Synthesis Inhibitor Prevent As2O3-InducedG2/M Arrest and Apoptosis

Apoptosis is an active progress that requires genexpression and protein synthesis. Inhibition of genexpression or protein synthesis can prevent apoptosis.o investigate whether a protein synthesis inhibitorould prevent As2O3-induced G2/M arrest and apopto-

is, exponentially growing cells were treated for 24 hith 4 mM As2O3 alone or in the presence of 20 mg/ml

ycloheximide (CHX). As shown in Fig. 5A, about 77%f the cells had arrested at G2/M phase after beingreated with As2O3 alone and only about 12% of theells treated with cycloheximide alone or cycloheximideombination with As2O3. Western blot analysis re-ealed that cycloheximide did not affect cdk2 and cdc2xpression, however, cyclin A, B and caspase-3 wereown-regulated by cycloheximide alone. The apoptosis

ragmentation induced by As2O3. Apoptosis was analyzed by flows and bars represent standard deviations. Fragmented DNA wastosis regulatory protein expression levels. Cells were treated with(50 mg) were subjected to electrophoresis and analyzed by Westerntreated with various concentrations of As2O3 for 24 h and harvestedubation of 20 mg of total protein with 200 mM chromogenic substrateophore p-nitroanilide (pNA) was monitored spectrophoto metricallynd bars represent standard deviations.

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roportion of cells treated with As2O3 plus cyclohexi-ide was reduced compared to that of cells treatedith As2O3 alone (Fig. 5A). We found that 20 mg/ml

ycloheximide alone also was able to induce some apop-osis. Western blot analysis revealed that cyclohexi-ide plus As2O3 did not affect cdc2 expression levels,

ut cycloheximide prevented cyclin B accumulationnd bcl-2 phosphorylation (Fig. 5B). Subsequent West-rn blotting demonstrated proteolytic cleavage ofLC-g1 in U937 cells after 24 h of 4 mM As2O3. Theleavage of PLC-g1 was significantly decrease in cellsreated with As2O3 plus cycloheximide (Fig. 5C). Tourther investigate and quantitate the proteolytic ac-ivity of caspase-3, we performed an in vitro assayased on the proteolytic cleavage of DEVD-pN. Theaspase-3 activity of cells treated with As2O3 plus cy-loheximide was reduced compared to that of cellsreated with As2O3 alone (Fig. 5D).

ffects of Aphidicolin on As2O3-InducedG2/M Arrest and Apoptosis

To investigate whether apoptosis was a consequencef the G2/M arrest, cells were treated for 12 h with 0.5g/ml aphidicolin prior to the addition of 4 mM As2O3.he effect of aphidicolin pre-treatment on the mediatedpoptosis was studied by FACS analysis. Cells treatedith 0.5 mg/ml aphidicolin for 24 h arrested in S phase.s shown in Fig. 6A, G2/M phase arrest and apoptosis

n cells treated with As2O3 plus aphidicolin was signif-

FIG. 3. Kinetic analysis of As2O3 mediated G2/M phase arrest antreated with 4 mM As2O3 for the indicated periods of time. Data artandard deviations. (B) Effect of As2O3 on cell cycle and apoptosiss2O3 for the indicated periods of time. Equal amounts of whole cestern blot for cdk2, cdc2, cyclin A, cyclin B, bcl-2, caspase-3, and

nd apoptosis. Cell cycle and apoptosis analysis of U937 cells treated ore mean values from three independent experiments and bars representregulatory protein expression levels. Cells were incubated with 4 mMell lysates (50 mg) were subjected to electrophoresis and analyzed byPARP.

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FIG. 4. Caspase-mediated apoptosis induced by Arsenic trioxide.A) Effects of z-VAD-fmk on 4 mM As2O3-induced caspase-3 activa-ion. U937 cells were incubated with z-VAD-fmk or solvent for 1 hefore treatment with 4 mM As2O3. Caspase-3 activity was deter-ined as shown in the figure. Data are mean values from three

ndependent experiments and bars represent standard deviations.B) Effects of z-VAD-fmk on cleavage of PLC-g1. U937 cells werencubated with z-VAD-fmk or solvent for 1 h before treatment with 4M As2O3. Equal amounts of cell lysates (40 mg) were subjected tolectrophoresis and analyzed by Western blot for caspase-3 andLC-g1. The proteolytic cleavage of PLC-g1 was indicated by arrow.

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cantly reduced compared to cells treated with As2O3

lone. To examine whether bcl-2 phosphorylation wasnvolved in As2O3 plus aphidicolin treated cells, weooked at bcl-2 phosphorylation and proenzyme levelsf caspase-3 by Western blot. As shown in Fig. 6B,yclin A and cyclin B were up-regulated in aphidicolinreated cells, but bcl-2 phosphorylation was not de-ected in the As2O3 plus aphidicolin treated cells.aken together, our results demonstrate that As2O3

auses the activation of cyclin B-dependent kinase andhe accumulation of cyclin B in association with mitoticrrest. G2/M arrest thus precedes the induction ofpoptosis.

ISCUSSION

In the present study we demonstrate that As2O3

nduces a marked increase in cyclin B accumulation

FIG. 5. Effect of cycloheximide on As2O3-induced G2/M phase arresence of 4 mM As2O3, 20 mg/ml cycloheximide alone, and cyclohexytometer. Data are mean values from three independent experimenn As2O3-induced G2/M phase arrest and apoptosis-related regulatorere subjected to electrophoresis and analyzed by Western blot for c

leavage of PLC-g1. Equal amounts of cell lysates (40 mg) were subjeLC-g1. The proteolytic cleavage of PLC-g1 was indicated by arrow. (Daspase-3 activity was determined as shown in Fig. 4. Data are mtandard deviations.

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nd induces the activation of cdc2 kinase in U937 cells.his occurs in a dose- and time-dependent manner inarallel with As2O3 induced G2/M phase arrest. Thenduction of apoptosis by As2O3 involves bcl-2 phos-horylation, caspase-3 activation, and cleavage ofARP and PLC-g1. As2O3 induced G2/M phase arrestas correlated with apoptosis. We evaluated how G2/Mrrest was linked to apoptosis, and our cycloheximidend aphidicolin results suggest that G2/M arrest trig-ers an apoptosis signal that leads to bcl-2 phos-horylation.When As2O3 was used to treat different types of

eukemia, consistent responses obtained only in APL3). In terms of a mechanism for the effect of on APLells, it has been suggested based on experiments withB4 cells of APL that arsenic caused apoptosis directly

hrough down-regulation of bcl-2 (11, 12). However, inontrast to previous reports, we did not observe bcl-2

and apoptosis. (A) U937 cells were incubated in the absence or theide plus As2O3. After a 24-h incubation, cells were analyzed by flowand bars represent standard deviations. (B) Effect of cycloheximiderotein expression levels. Equal amounts of whole cell lysates (50 mg), cdc2, cyclin A, cyclin B, and bcl-2. (C) Effects of cycloheximide on

d to electrophoresis and analyzed by Western blot for caspase-3 andffects of cycloheximide on 4 mM As2O3-induced caspase-3 activation.

n values from three independent experiments and bars represent

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own-regulation in U937 cells treated with 6 mMs2O3. This result is consistent with a recent reporthowing that bcl-2 was not down-regulated in As2O3-reated HL-60 cells (10). One proposal of a molecularechanism for As2O3-induced apoptosis was explained

y glutathione redox system. Cells exhibiting low ac-ivities of GST p, catalase, and glutathione peroxideGPx) were more sensitive to As2O3 induced apoptosis9, 10). However, this was not fit in K562 cells andome other cells. To date, the molecular mechanism ofhe G2/M arrest and the apoptosis induced by As2O3

emains unclear. We demonstrated that the G2/Mhase arrest is observed concomitantly with the onsetf apoptosis. The As2O3 induced apoptosis signalingathway involved bcl-2 phosphorylation, caspase-3 ac-ivation, and PARP cleavage. We first showed thats2O3 induced bcl-2 phosphorylation in U937 cells.hosphorylation of bcl-2 leads to the dissociation ofcl-2/bax heterodimers inducing the formation of bax/ax homodimers which promote cytochrome c release

FIG. 6. Effect of aphidicolin on As2O3-induced G2/M phase arresphidicolin and then with 4 mM As2O3 for 24 h. Cells were harvestndependent experiments and bars represent standard deviationspoptosis-related regulatory protein expression levels. Equal amounnalyzed by Western blot for cdk2, cdc2, cyclin A, cyclin B, bcl-2, an

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rom mitochondria (22–24). Cytochrome c release haseen seen to stimulate an apoptotic protease cascadenvolving caspase (27). Furthermore, induction of bcl-2hosphorylation has been observed in various cellypes exposed to taxol and related agents that interfereith microtubule dynamics (23, 28). Li et al. recently

eported that the effect of the As2O3-induced mitoticrrest is very similar to the effect of antitubulin drugs17). However, taxol-induced bcl-2 phosphorylation haseen shown to be dependent on Raf-1, while As2O3

ediated bcl-2 phosphorylation seems not to be linkedo Raf-1 activation, since we did not see any Raf-1ctivation in As2O3 treated cells (data not shown, 29, 30).It has been recently reported that the clinical remis-

ion induced by As2O3 in APL patients is associatedith incomplete differentiation and the induction ofpoptosis via caspase-1 and -3 activation (3). Caspase-3s a key apoptosis regulatory molecule that becomesctivated during the early stages of apoptosis (21).ctive caspase-3 consists of a heterodimer of 17 and 12

d apoptosis. (A) U937 cells were pretreated for 12 h with 0.5 mg/mland analyzed by flow cytometer. Data are mean values from three) Effect of aphidicolin on As2O3-induced G2/M phase arrest andof whole cell lysates (50 mg) were subjected to electrophoresis andaspase-3.

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Da subunits which are derived from a 32 kDa proen-yme. The activated caspase-3 in As2O3 treated cells isccompanied by cleavage of PARP and PLC-g1.In the present study, we explored the functional

elationship between G2/M arrest and apoptosis ins2O3 induced U937 cells. The progression from G2

nto mitosis is dependent on the modulation of cdc2 (13,4). The regulation of cdc2 was associated with cyclinartners and phosphorylation status. The As2O3 medi-ted apoptosis that we observed after activation of cdc2s consistent with several reports that suggest a linketween activation of cdc2 and apoptosis. Shi et al.eported that apoptosis was prevented by blocking cdc2ctivity with excess peptide substrate, or by inactivat-ng cdc2 in temperature-sensitive mutant (31). Simi-arly, activation of the cdc2 by taxol, Fas receptor,GF-b and DNA damage at an inappropriate timeuring the cell cycle also leads to apoptosis (15, 16,2–34). Park et al. recently reported that As2O3-ediated cell cycle arrest in G1 and G2/M phase was

ssociated with enhanced p21 binding activity withdk6, cdc2, cyclin E and cyclin A in Myeloma cells. Inddition, the activity of cdc2-associated kinase wasecreased after their exposure to As2O3 (35). Theseesults were not consistent with our data. The discrep-ncy between cdc2-associated kinase activity underhese conditions may be related to the different cellines, dose and time of As2O3 exposure. Our resultsemonstrate that As2O3 induces cyclin B expressionnd activation of its kinase in parallel with G2/Mhase arrest. To investigate whether apoptosis was aonsequence of the G2/M arrest, we performed twoxperiments. First, we analyzed As2O3-induced apopto-is in cells pretreated with aphidicolin, which inhibitsNA polymerase. As2O3 did not induced apoptosishen the G2/M phase arrest was prevented by aphidi-

olin. Second, we used the protein inhibitor cyclohexi-ide. Cycloheximide by itself induced apoptosis, but

ycloheximide plus As2O3 did not enhance apoptosisnd G2/M arrest. Therefore, As2O3 induced apoptosisequires protein synthesis and in the presence of pre-ious G2/M arrest. Our data raise the possibility thatrsenic trioxide could be used to lower the apoptotichreshold of leukemia cells and thus potentially im-rove the treatment of human acute promyelocyticeukemia.

CKNOWLEDGMENT

This work was supported by Grant 2000-1-20700-005-3 fromhe Basic Research Program of the Korea Science and Engineeringoundation.

EFERENCES

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Arsenic Trioxide Induces G2/M Growth Arrest and Apoptosis after Caspase-3 Activation and Bcl-2...

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