(CANCER RESEARCH S3. 3<í7fc-3<W5.Seplember 1, 1993]

Specific Proteolytic Cleavage of Poly(ADP-ribose) Polymerase: An Early Marker ofChemotherapy-induced Apoptosis1

Scott H. Kaufmann,2 Serge Desnoyers, Yvonne Ottaviano, Nancy E. Davidson, and Guy G. Poirier

The Oncology Center, Johns Hopkins Hospital (S. H. K., Y. O., N. E. D.), and Department of Pharmacology (S. H. K.), The Johns Hopkins University School of Medicine,Baltimore, Maryland 212N7, and PolytADP-Ribose) Metabolism Croup, Department of Molecular Endocrinology; CeñiréHospitalier de l'Université Laval Research Center and

Laval University (S. D., G. G. P.), Sainte-Foy; Québec,Canada G1V4G2

ABSTRACT

Apoptosis ¡sa morphologically and biochemically distinct form of celldeath that occurs under a variety of physiological and pathological conditions. In the present study, the proteolytic cleavage of poly(ADP-ribose)polymerase (pADPRp) during the course of chemotherapy-induced apo-ptosis was examined. Treatment of HL-60 human leukemia cells with thetopoisomerase H-directed anticancer agent etoposide resulted in morpho

logical changes characteristic of apoptosis. Endonucleolytic degradation ofDNA to generate nucleosomal fragments occurred simultaneously. Western blotting with epitope-specific monoclonal and polyclonal antibodies

revealed that these characteristic apoptotic changes were accompanied byearly, quantitative cleavage of the U, 116,000 pADPRp polypeptide to anU, —¿�25,0110fragment containing the amino-terminal DNA-binding domain of pADPRp and an \t, —¿�85,000fragment containing the automod

ification and catalytic domains. Activity blotting revealed that the U,—¿�85,000fragment retained basal pADPRp activity but was not activated

by exogenous nicked DNA. Similar cleavage of pADPRp was observedafter exposure of HL-60 cells to a variety of chemotherapeutic agentsincluding m-diaminedichloroplatinum(II), colcemid, 1-ß-n-arabinofura-nosylcytosine, and methotrexate; to y-irradiation; or to the protein syn

thesis inhibitors puromycin or cycloheximide. Similar changes were observed in MDA-MB-468 human breast cancer cells treated withtrifluorothymidine or 5-fluoro-2'-deoxyuridine and in y-irradiated or glu-

cocorticoid-treated rat thymocytes undergoing apoptosis. Treatment withseveral compounds (tosyl-i.-lysine chloromethyl ketone, tosyl-i.-phenylala-nine chloromethyl ketone, /V-ethylmaleimide, iodoacetamide) prevented

both the proteolytic cleavage of pADPRp and the internucleosomal fragmentation of DNA. The results suggest that proteolytic cleavage of pAD-PRp, in addition to being an early marker of chemotherapy-induced

apoptosis, might reflect more widespread proteolysis that is a criticalbiochemical event early during the process of physiological cell death.

INTRODUCTION

The term apoptosis denotes a distinctive sequence of morphologicalchanges observed in certain cells as they die (reviewed in Refs. 1-6).

In the nucleus, these morphological changes include condensation ofthe nuclear contents into clumps of heterochromatin adjacent to thenuclear envelope, then nuclear fragmentation, and finally packagingof the nuclear fragments into multiple membrane-enclosed apoptotic

bodies. These morphological changes have been observed in cells thatdie during morphogenesis (reviewed in Refs. 1 and 2), after withdrawal of trophic hormones (7-11), and after exposure of cells tochemotherapeutic agents (1, 12-15), y-irradiation, or cytotoxic lym

phocytes (reviewed in Refs. 1, 3, 6, and 16). Conversely, it has beenobserved that treatment of cells with tumor-promoting phorbol esters

Received 3/3/93; acceplcd 6/21/93.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.

1Supported in part by grants from the NIH (CA50435. CA55642, CA57545), Canadian

Medical Research Council (MTM28), and Natural Sciences and Engineering ResearchCouncil of Canada (AO415). S. H. K. was supported by a Clinical Oncology CareerDevelopment Award from the American Cancer Society. S. D. received a predoctoralfellowship from the Fonds pour la Formation de Chercheurs et l'Aide àla Recherche.

Y. O. was supported by a Steiler Research Fund fellowship.2 To whom reprint requests should be addressed, at Oncology 1-127. Johns Hopkins

Hospital. 600 N. Wolfe Street, Baltimore, MD 21287.

(17-19) or overexpression of the bcl-2 oncogene (20-23) can inhibit

the events leading to apoptosis in some cell systems. Understandingapoptotic cell death has, therefore, assumed an increasingly important role in the fields of experimental oncology and developmentaltherapeutics.

The morphological alterations of apoptosis are accompanied by avariety of biochemical changes. Elevations in cytosolic free calcium(reviewed in Ref. 24) and cytosolic hydrogen ion (25) are followed byinternucleosomal DNA degradation (reviewed in Refs. 5, 16, 26, and27) and sharp decreases in cellular NAD levels (28-31). These

changes typically occur at the time that cells lose their proliferativecapacity but before cells become permeable to dyes such as propidiumiodide or trypan blue.

Although previous experiments have focused extensively on theactivation of an endonuclease that catalyzes internucleosomal DNAdegradation (reviewed in Refs. 5, 16, 26, and 27; see also 32-34),activation of the nuclear enzyme pADPRp1 is also thought to play a

role in the biochemical changes that accompany apoptosis (reviewedin Refs. 35-38). pADPRp is a Mr 116,000 polypeptide that is present in ~106 copies/cell in somatic cells (39, 40). This enzyme con

verts NAD to nicotinamide and protein-linked ADP-ribose polymers

(reviewed in Refs. 41 and 42). Results of studies of endogenousADP-ribose polymer levels suggest that the polymerase is relativelyinactive in unperturbed tissue culture cells (43-45). Polymer levels

increase markedly after treatment of cells with agents that induceDNA single-strand and double-strand breaks (43-45), presumably as

a consequence of pADPRp activation (46, 47) that results from aconformational change when the enzyme binds to damaged DNA(48) through one of its two zinc fingers (reviewed in Ref. 49). Activated pADPRp catalyzes the poly(ADP-ribosyl)ation of a variety of

nuclear polypeptides, but most of the polymer chains are bound topADPRp itself (reviewed in Refs. 41 and 42). It has recently beenshown in human cell extracts that this automodification of pADPRpfacilitates release of the protein from damaged DNA and permits access of DNA repair enzymes to DNA single-strand breaks blocked

by unmodified pADPRp (50).Several investigators (29-31, 51) have proposed that the activation

of pADPRp by DNA strand breaks contributes to the consumption ofNAD that occurs in cells undergoing apoptosis. On the other hand.Nelipovich et al. (52) have reported that pADPRp activity actuallydecreases during the course of radiation-induced apoptosis in rat thy

mocytes. Additional studies using enzyme inhibitors have failed toclarify the role of pADPRp in apoptosis. Treatment with millimolarconcentrations of 3-AB, an inhibitor of NAD-requiring enzymes, delays apoptosis in certain cell systems (30, 51, 53-55). These results

have been interpreted as indicating that activation of pADPRp contributes to apoptosis. More recent studies, however, indicate that the3-AB concentrations utilized in these studies could potentially inhibit

other enzymes (56). To add to the confusion, other inhibitors of

1 The abbreviations used are: pADPRp, poly(ADP-ribose) polymerase; 3-AB.3-aminobenzamide; E-64, (W[/V-L-3-/rarts-carboxyoxiran-2-carbonyl)-i-leucyl]-agmatine;NAD. nicotinamide adeninc dinucleotide; SDS, sodium dodecyl sulfate; TLCK, tosyl-L-lysine chloromethyl ketone; PBS, phosphate-buffered saline; PAGE, polyacrylamide gelelectrophoresis; cytosine arabinoside, 1-ß-u-arabinofuranosylcytosine.

3976

CLEAVAGE OF POLY(ADP-RIBOSE) POLYMERASE

pADPRp have been observed to provoke apoptosis, leading to thehypothesis that pADPRp activity normally protects the cell fromapoptosis (57). Thus, the role of pADPRp in cells that undergo apoptosis is unclear.

It has been generally accepted that the macromolecular degradationin cells undergoing apoptosis is initially limited to internucleosomalDNA degradation. A previous study from this laboratory, however,revealed that internucleosomal degradation of DNA in etoposide-treated HL-60 human leukemia cells was accompanied by the early,

quantitative cleavage of the M, 116,000 pADPRp polypeptide to arelatively stable MT —¿�85,000fragment (58). A similar cleavage of

pADPRp was observed in Molt4 cells undergoing purineless celldeath after treatment with the glycineamide ribonucleotide trans-formylase inhibitor 5-deazaacyclotetrahydrofolate (59). It was unclear

whether this cleavage of pADPRp was limited to human leukemia celllines treated with a select group of agents or whether it was a generalphenomenon occurring in a wide variety of cell types undergoingchemotherapy-induced apoptosis. Moreover, the effect of this cleav

age event on the catalytic activity of pADPRp was unknown. In thepresent study, the nature of this proteolytic cleavage and its effect onpADPRp activity have been studied in relation to changes in NADlevels in etoposide-treated HL-60 cells. In addition, a number of

different experimental systems that undergo apoptosis were examinedfor pADPRp cleavage. Finally, to clarify the importance of this earlyproteolysis, the effect of protease inhibitors on the fragmentation ofpADPRp and DNA was examined.

MATERIALS AND METHODS

Materials. Etoposide was kindly provided by Bristol-Myers (Syracuse,NY). Phosphoramidone and E-64 were from Boehringer-Mannheim (Indianapolis, IN). Puromycin, cytosine arabinoside, methotrexate, cv'.v-platinum, 3-AB,

trifluorothymidine, 5-fluoro-2'-deoxouridine, bisbenzimide, and the remaining

protease inhibitors were obtained from Sigma (St. Louis, MO). 32P-labeled

NAD and nitrocellulose for activity blots were from New England Nuclear(Boston, MA) and Amersham (Oakville, Ontario), respectively. Sources of allother reagents have been previously described (58).

Buffers. Medium A consisted of RPMI 1640, 10% (v/v) heat-inactivated

fetal bovine serum, 100 (xg/ml streptomycin, 100 fig/ml penicillin G. and 2 HIMglutamine. Buffer B consisted of 6 M urea, 2% (w/v) SDS. 62.5 ITIMTris-HCl(pH 6.8), 0.15 M/3-mercaptoethanol, and 0.1% (w/v) bromphenol blue. BufferC consisted of 6 M guanidine hydrochloride, 250 HIMTris-HCl (pH 8.5 at21°C), 10 HIMEDTA, and 1% (v/v) ß-mercaptoethanol. Immediately before

use of each aliquot of buffer C, a-phenylmethylsulfonyl fluoride was added to

a final concentration of 1 min from a 100 ITIMstock in anhydrous isopropanol.Buffer D consisted of 0.7 M ß-mercaptoethanol, 190 ITIMglycine, 25 HIMTris-HCl (pH 8.0), and 0.1% (w/v) SDS. Buffer E consisted of 50 mMTris-HCl

(pH 8.0), 100 rriM NaCl, 1 mM dithiothreito!, 20 JAMZn(II) acetate, 2 ITIMMgCl2, and 0.3% (v/v) Tween-20. Buffer F consisted of buffer E without Zn(II)

acetate and MgCl2. PBS contained 137 mw NaCl, 2.7 mm KC1, 1.5 mMKH2PO4, and 8.0 min Na2HPO4.

Drug Treatment and Irradiation. Logarithmically growing human HL-60

progranulocytic leukemia cells or K562 chronic myelogenous leukemia cells(propagated at densities of < 1 X 106/ml in medium A) were freed of senescent

cells by sedimentation over a Ficoll-Hypaque step gradient (density = 1.119g/cm3) and resuspended at a concentration of 1—1.5X 10'Vml in fresh medium

A. Etoposide was added from a 1000-fold concentrated stock in dimethylsulfoxide. After various periods of incubation at 37°Cin an atmosphere con

taining 5% (v/v) CO2:95% air, cells were sedimented at 3200 X g for 1 min andlysed in SDS-proteinase K in preparation for agarose gel electrophoresis (59).

For Western blots, cells were sedimented at 200 X g for 10 min, washed oncein serum-free RPMI 1640 containing 10 mM 4-(2-hydroxyethyl)-l-pipera-zineethanesulfonic acid (pH 7.4 at 21 °C),and lysed by sonication in buffer C

(60). For activity blots, drug-treated cells were washed as described for West

ern blotting and solubilized by vigorous vortexing for 1 min in buffer B at aconcentration of 4 x IO7 cells/ml. Cells for NAD levels were sedimented at400 X g for 10 min, washed twice at 4°Cwith PBS, and extracted for 15 min

on ice with 0.5 M perchloric acid. After the extracts were neutralized with 0.5volume of 1 M KOH containing 0.2 M potassium phosphate (pH 7.5 afterneutralization), NAD was determined using an enzymatic cycling assay exactlyas described by Bernofsky and Swan (61). Cells for morphological examination were washed twice with PBS, fixed in 3:1 (v:v) methanol:acetic acid,pipetted dropwise onto glass slides, rehydrated with PBS, stained with 1 ng/mlbisbenzimide in 50% glycerol, and examined by fluorescence microscopyusing a Zeiss universal microscope equipped with an epi-illuminator and

appropriate filters.In some experiments, etoposide was replaced with cytosine arabinoside,

methotrexate, or cis-platinum (prepared as 1000-fold concentrated stocks indimethyl sulfoxide). Alternatively, HL-60 cells were treated with puromycin or

cycloheximide (added from 1 mM stocks in tissue culture medium) or wereirradiated at 1.0 Gy/min to a final dose of 10 Gy using a '"Co source.

MDA-MB-468 cells (kindly provided by Dr. Marc Lippman, Georgetown

University, Washington. DC) were exposed to 100 /MMtrifluorothymidine or5-fluoro-2'-deoxyuridine (prepared as filter-sterilized 10 mM aqueous stocksthat were frozen at -20°C until use). Adherent and nonadherent cells were

either harvested separately as previously described (14) or combined, washedwith serum-free medium, and soluhilized in buffer C as described above.

Rat thymocytes (isolated as described in Ref. 62) were incubated at 37°Cin

serum-free RPMI 1640 medium containing 10 mM 4-(2-hydroxyethyl)-l-pip-erazineethanesulfonic acid (pH 7.4) in the presence or absence of l (J.Mdexa-methasone (added from a 1000-fold concentrated ethanolic stock). Alternatively, thymocytes were irradiated to 6.0 Gy from a 137Cs source at a dose of

1.0 Gy/min prior to incubation in the absence of dexamethasone. Analyses ofprotein and DNA were performed as described above except that cells weresedimented at 12,000 x g for 2 min prior to lysis.

Western Blotting. After reduction, alkylation with iodoacetamide, andsubsequent preparation for electrophoresis (60), samples containing 50 jj.g ofprotein (MDA-MB-468 cells, thymocytes) or protein from 3 X HP leukemiacells were applied to SDS gels prepared with a 5-15% (w/v) polyacrylamide

gradient. After electrophoretic transfer of the separated polypeptides to nitrocellulose, Western blotting was performed as previously described (58). Theepitopes recognized by the various antibodies are indicated in Fig. 2F. C-2-10

is a mouse monoclonal antibody that recognizes an epitope located at thecarboxyl end of the DNA-binding domain (63). Anti-F2 (generously provided

by Dr. G. deMurcia, Centre National de la Recherche Scientifique, StrasbourgCedex, France) is a rabbit polyclonal antiserum raised against a syntheticpolypeptide corresponding to the second zinc finger of human pADPRp (64).

Activity Blotting. Electrophoretically separated polypeptides were assayedfor pADPRp activity as described by Simonin et al. (65). Briefly, samplescontaining protein from 4 x IO5 cells were applied to adjacent wells of

SDS-containing minigels prepared with 8% (w/v) polyacrylamide. After electrophoresis at 200 V, gels were soaked for 60 min at 37°Cin buffer D and

electrophoretically transferred to nitrocellulose. Immobilized polypeptideswere renatured for l h in buffer E, incubated with 2 fiCi/ml 3-P-labeled NAD

in buffer E (with or without I fig/ml of DNase-activated DNA), and washedwith isotope-free buffer F.

RESULTS

Etoposide-induced Apoptosis in HL-60 Cells. Treatment ofHL-60 human progranulocytic leukemia cells with the topoisomeraseIl-directed chemotherapeutic agent etoposide resulted in morpholog

ical changes consistent with the process of apoptosis (Fig. 1). Initiallya rim of heterochromatin appeared at the nuclear periphery (Fig. Iß).The nucleolus simultaneously disappeared. The nuclei subsequentlyfragmented, and each cell formed multiple membrane-enclosed vesi

cles (Fig. 1C; see also Fig. 9C). As is the case in other cell systemsundergoing apoptosis, mitochondria and the plasma membrane remained intact throughout the course of these morphological changes.Consequently, —¿�95%of the cells continued to exclude trypan blue

after 6 h.These morphological changes were accompanied by progressive

internucleosomal degradation of DNA to yield a ladder of DNA fragments (Fig. 24) as previously reported by this laboratory and others(15, 58, 66-68). When the proteins in these etoposide-treated cells

3977

CLEAVAGE OF POLY(ADP-RIBOSE) POLYMERASE

Oh

B 2h

cleavage of pADPRp was observed at etoposide concentrations as lowas 4 /¿M(data not shown).

Location of the Cleavage Site in pADPRp. To assess the site ofcleavage of pADPRp, Western blotting was repeated with anti-F2, a

polyclonal antibody raised against the second zinc finger of pADPRp(Fig. 2F). During the course of etoposide treatment, loss of the M,116,000 polymerase (Fig 2D, lane 1) was accompanied by the appearance of an Mr —¿�25,000polypeptide recognized by this antibody

Oh 1 1.5 2 3

200

».

6hFig. 1. Etoposide induces typical apoptotic morphological changes in HL-6Ãœcells.

HL-60 cells were incubated with 68 |UMetoposide for 0 (A). 2 (B), or 6 h (C). Sampleswere fixed and prepared for electron microscopy as previously described (59). A nuclearrim of heterochromatin was evident within 2 h (B). By 6 h, the nuclei were fragmentedand pyknotic (C). These changes were observed in >80% of cells. Even after 6 h, theplasma membrane remained intact, and the mitochondria were morphologically normal,findings that are consistent with the observation that >95% of cells continue to excludetrypan blue at 6 h. Similar changes were observed when HL-60 cells were treated withetoposide for I h and subsequently incubated in the absence of drug.

were separated by SDS-PAGE and stained with Coomassie blue, the

bulk of the cellular polypeptides remained intact (Fig. 25). However,Western blotting with monoclonal antibody C-2-10, an antibody thatrecognizes an epitope at the carboxyl end of the DNA-binding domain

of pADPRp (Fig. 2F), revealed that the Mr 116,000 pADPRp molecule was degraded to a relatively stable Mr —¿�85,000fragment with a

time course that paralleled the DNA fragmentation (Fig. 2C). Similar

-57

C-2-1057-

-31

EFF1F2Jü1

!anti-F2Anti-B23

DAnti-FP

CtrIiîc"46kDiDNA-b22

kM.iHUta„din,

Alicorno- NAD.bindiri{

-13

Fig. 2. Proteolytic cleavage of pADPRp accompanies endonucleolylic DNA degradation in ctoposide-treated HL-60 cells. HL-60 cells were treated with 68 ¿LMetoposide forO, I. 1.5, 2, 3, or 4 h (lanes 1-6, respectively). Aliquots were prepared for agarose gelelectrophoresis (A) or SDS-PAGE (B-E). One gel was stained with Coomassie blue (B).

Companion gels were transferred to nitrocellulose and reacted with monoclonal antibodyC-2-10 (C), polyclonal anti-F2 (D), or antibody against the nucleolar protein B23 (£).followed by their respective radiolabeled secondary antibodies. F, schematic of pADPRpindicating known cleavage sites for trypsin (t), papain (P), and chymotrypsin (C) as wellas the two zinc fingers (Fl and F2) and the location of epitopes recognized by C-2-10 andanti-F2. Lower schematic indicates functional domains of pADPRp (*í8).Numbers al left

and righi, sizes of standard DNA fragments in kilobases or polypeptides in thousands.

3978

CLEAVAGE OF POLYlADP-R1BOSE) POLYMERASE

-DMA +DNAOh 1 1.5 Oh 1 1.5 2

34

B

116-85-

123 456

116-

Fig. 3. Activity blot of pADPRp performed in the absence (A and C) or presence (B andD) of exogenous nicked DNA. HL-60 cells treated with 68 ^IMetoposide for 0, I, 1.5. 2,3, or 4 h (lanes 1-6, respectively) were subjected to SDS-PAGE. After polypeptides weretransferred to nitrocellulose and renatured in situ as described in the "Materials andMethods," blots were incubated with ';P-labeled NAD in the absence (A and C) or

presence (B and D} of exogenous nicked DNA. In A and B. simultaneously preparedautoradiogrephs demonstrate that the M, ~85.<MH)fragment is labeled by '-P-NAD in the

absence of exogenous DNA (e.g.. A, lane 6} but is less strongly labeled in the presenceof exogenous DNA (e.g.. B. lane 6ì.C and /). simultaneously prepared autoradiographswere underexposed to demonstrate the activation of the M, 116.0ÕX)polymerase byexogenous nicked DNA (D) compared to baseline activity (C). £,glycohydrolase digestion to confirm that bound radiolabel was incorporated into polvi ADP-ribose) polymer.Lanes !-.<, samples from HL-60 cells treated with 68 IHMetoposide for 0. I. or 2 h.respectively. Lanes 4-6, after autoradiography. the samples in lanes I and 2 were incubated with buffer containing (lane 4) or lacking (lune 5} glycohydrolase. Auloradiographyrevealed that glycohydrolase completely removed the bound radiolabel (lane 4 ). The pieceof nitrocellulose corresponding to lane 5 was inadvertently reversed during remounting(cf. lanes 2 and 5). Ordinale. M, in thousands.

(Fig 2D, lanes 4-6). These results suggested that pADPRp wascleaved to fragments of M, —¿�85.000(lacking the zinc finger domain)and MT —¿�25.000(containing the zinc finger domain). In contrast, the

nucleolar polypeptide B23/nucleophosmin, which has a nuclear localization similar to pADPRp (69). remained intact during etoposide-

induced apoptosis (Fig. 2E).Afr 85,000 Fragment Retains DNA-insensitive Catalytic Ac

tivity. If pADPRp were fragmented without significant proteolysis atits carboxyl terminus, the M, —¿�85.000fragment described above

would be expected to retain enzyme activity (64). Moreover, removalof the zinc fingers would be expected to render the M, —¿�85.000

fragment insensitive to activation by the addition of exogenous nickedDNA (64). To confirm these predictions, polypeptides from controland etoposide-treated HL-60 cells were separated by SDS-PAGE.

transferred to nitrocellulose, renatured. and assayed for activity in situby incubating the blots with 32P-labeled NAD as previously described

by Simonin el al. (65).When control samples were analyzed in this fashion, an Mr 116.000

polypeptide (the automodified pADPRp) became 12P-labeled (Fig.

3/4, lane I ). The bound radiolabel was not removed by subsequentincubation of the blots with 3% SDS (data not shown), a treatment thatremoves noncovalently bound radiolabel.4 In contrast, the radiolabel

was removed by incubation with purified glycohydrolase. an enzymethat selectively degrades poly(ADP-ribose) polymers (Fig. 3E). Ad

ditional experiments (not shown) revealed that the radiolabel couldalso be released from the immobilized Mr 116.000 polypeptide byNaOH treatment. Electrophoresis of this NaOH-released product in209Õ-(w/v) polyacrylamide gels (44) revealed a ladder of polymer

sizes characteristic of poly(ADP-ribose).4 Collectively, these results

indicate that the radiolabel bound to the MT 116.000 polypeptiderepresents poly(ADP-ribose) polymers that have become covalently

attached to the enzyme as a consequence of automodification.When extracts from etoposide-treated cells were analyzed, a

polypeptide of M, ~85,000 also became radiolabeled (Fig. 3/4, lanes4-6). Although it was not possible to isolate enough radiolabeled

material for size determination, control experiments revealed that theradiolabel remained bound to the M, —¿�85,000band after treatment

with 3% SDS but was removed by incubation with purified glycohydrolase.4 Thus, the Mr -85.000 polypeptide. like the intact Mr

116.000 polymerase, appears to be covalently modified by an NADderivative that is degraded by an enzyme that selectively degradespoly(ADP-ribose). These observations are consistent with the viewthat the MT —¿�85.000pADPRp fragment contains functional catalytic

and automodification domains (Fig. 2F). Furthermore, since removalof as few as 45 amino acids from the carboxyl terminus of pADPRpabolishes pADPRp activity (64), these results suggest that the carboxyl terminus of pADPRp is largely or completely intact in theMr -85,000 fragment.

When the activity blotting was repeated in the presence of exogenous nicked DNA. labeling of the M, 116,000 polypeptide was markedly increased (cf. C and D, Fig. 3). In contrast, labeling of the Mr—¿�85,000fragment was not enhanced and was probably diminished(cf. A and B, Fig. 3). Thus, the M, -85,000 fragment exhibits basal

catalytic activity, but its activity under conditions of maximal stimulation appears to be much lower than that of the intact polymerase.

Relationship of pADPRp Cleavage to Diminished NAD Levels.The decline of NAD levels observed in cells undergoing apoptosis hasbeen attributed to increased NAD consumption as a consequence ofpADPRp activation by internucleosomal DNA strand breaks (see "Introduction"). On the other hand, the cleavage of pADPRp described

above might be expected to diminish the cellular activity of pADPRp.In order to reconcile the consumption of NAD and the cleavage ofpADPRp. we examined NAD levels and pADPRp fragmentation inHL-60 cells treated with etoposide in the absence and presence of thepADPRp inhibitor 3-AB. In the absence of 3-AB, NAD levels diminished to 70% of control levels 60-90 min after addition of etoposide.

to 30% of control levels after 2 h. and to 20% of control levels by 3h (Fig. 4C). Thus, the bulk of the cellular NAD was consumed priorto the cleavage of pADPRp (cf. B and C, Fig. 4, lanes 1-6).

When the same experiment was performed in the presence of 200/XM3-AB. a concentration that selectively inhibits pADPRp (56),

NAD levels remained at control levels for the first 2 h (Fig. 4C).Nonetheless, fragmentation of the cellular DNA and proteolytic cleavage of pADPRp became evident by 2 h (Fig. 4, lane 10) and proceeded at the same rate as in the absence of 3-AB. Although these

results implicate pADPRp in the consumption of NAD that occursearly in the course of etoposide-induced apoptosis (cf. lanes 4 and 10.

Fig. 4C). they also suggest that pADPRp activity and consumption ofNAD do not play an obligate role in DNA fragmentation or pADPRpcleavage.5

Cleavage of pADPRp after Treatment with Other CytotoxicAgents. To ascertain whether the cleavage of pADPRp was unique toetoposide-induced apoptosis. HL-60 cells were treated with a varietyof cytotoxic agents including cytosine arabinoside. methotrexate. cis-

diaminedichloroplatinum(II), and colcemid. Results obtained with cytosine arabinoside and colcemid are shown in Fig. 5. Despite the

4 S. Desnoyers and G. G. Poirier, unpublished observations

5 The decrease in NAD levels at late times observed in the presence of 3-AB in Fig.4C might reflect consumption by other enzymes that are not inhibited by 3-AB. Alternatively, it might reflect consumption by a small amount of pADPRp that remains intact(Fig. 3D. lane 61 and is massively stimulated by the accumulating strand breaks observedin Fig. 4.1.

3979

CLEAVAGE OF POLY(ADP-RIBOSE) POLYMERASE

-3AB + 3AB

Oh 1 1.5 2 0 1 1.5 2 3

Fig. 4. Temporal relationship among DNA fragmentation (A), pADPRp cleavage (B),and cellular NAD levels (har graph) in the absence (lanes 1-6) or presence (lanes 7-12)of 3-AB. HL-6Ãœcells were treated with 68 /ÃŒMetoposide for the indicated time in theabsence or presence of 2(10 fin 3-AB and analyzed as described in "Materials andMethods." NAD levels were expressed as % of control (0 h. -3-AB) levels.

diverse mechanisms for these agents, each induced internucleosomalDNA degradation (Fig. 5,^4 and C, and Ref. 58). Even though the timecourse for the appearance of DNA fragments varied (cf. Figs. 2A, 5A,and 5C), in each case the initial appearance of oligonucleosomal DNAfragments was accompanied by cleavage of pADPRp (Fig. 5, B and D,and data not shown). Thus, the cleavage of pADPRp was not uniqueto treatment with etoposide.

Appearance of the pADPRp Fragment in the Presence of Protein Synthesis Inhibitors. In certain cell types, generation of endo-

nucleolytic DNA damage is prevented by inhibitors of RNA andprotein synthesis (70-74). This has led to the concept of a genetic

program involved in cell death. In other cell types, however, the samemorphological changes and DNA degradation occur in the presence ofprotein synthesis inhibitors (58, 75-79). In the current study, HL-60

cells were incubated with puromycin at a concentration that inhibitedprotein synthesis by 95%. This treatment resulted in the appearance of

a nucleosomal DNA ladder (Fig. 6A) and degradation of pADPRp(Fig. 6B) within 2 h. Similar results were obtained with cyclohexim-ide (data not shown). Therefore, it appears that the cleavage of pAD-

PRp, like the endonucleolytic degradation of DNA, can occur despiteextensive inhibition of protein synthesis. These results raise the possibility that the endonuclease and protease detected in these assays arepreexisting cellular enzymes, although the selective synthesis of theendonuclease and protease in the presence of puromycin or cyclohex-imide also cannot be ruled out.

Cleavage of pADPRp in Other Cells Undergoing Apoptosis. Toexclude the possibility that the cleavage of pADPRp occurred only inHL-60 cells, the fragmentation of DNA and pADPRp were examinedin a variety of cell types undergoing chemotherapy-induced apoptosis.

In human KG la acute myelogenous leukemia cells as well as Molt 3acute lymphocytic leukemia cells treated with etoposide, internucleosomal DNA degradation was again accompanied by cleavage of pAD-

PRp (data not shown). In contrast, human K562 chronic myelogenousleukemia cells, a cell type previously reported to resist diphtheriatoxin-induced apoptosis (76), failed to show evidence of endonucle

olytic DNA degradation (Fig. 1A) and pADPRp cleavage (Fig. IB)after a 24- to 48-h treatment with lethal doses of etoposide. These

observations strengthen the association between endonucleolyticDNA degradation and pADPRp fragmentation.

To determine whether proteolytic cleavage of pADPRp occurredonly in leukemia cell lines, MDA-MB-468 human breast cancer cellswere treated for up to 48 h with trifluorothymidine or 5-fluoro-2'-

Ara-C

Colcemid3h 6 18

0.6-

116-

85-

116-

85-

B C-2-10

Fig. 5. cytosine arabinoside (Ara-C; A and B) or colcemid (C and D) induce DNAfragmentation (A and C) and cleavage of pADPRp (B and D). In A and B, HL-60 cellswere incubated with l ¡J.Mcytosine arabinoside for the indicated time (lanes 2-6) and thenanalyzed as described in "Materials and Methods." As controls, cells were incubated for

4 h in the presence (lane I) or absence (lane 7) of 68 /AMetoposide (VP). In C and D,HL-60 cells were incubated for the indicated time with 1 /Ag/ml coicemid.

3980

CLEAVAGE OF POLYlADP-RlBOSEl POLYMERASE

deoxyuridine. two pyrimidine analogues that result in thymidinelesscell death. Cleavage of pADPRp was evident in these cells at 24 h(Fig. 8/4). Previous results (14. 80) have revealed that apoptosis-

associated internucleosomal DNA cleavage in adherent cell lines ismost readily demonstrated in the cells that lift off the tissue cultureplates. Western blotting revealed that cleavage of pADPRp also occurred exclusively in the nonadherent cell population (Fig. SB). Furthermore, this analysis revealed that fragmentation of pADPRp occurred in the small population of cells that spontaneously underwentapoptosis in this cell culture (Fig. 8ß.Oh). Thus, cleavage of pADPRpis not a consequence of the drug treatment per se. Instead, this cleavage is a component of the apoptotic process.

Finally, to rule out the possibility that proteolytic cleavage of pAD-

PRp only occurred in neoplastically transformed cells, rat thymocyteswere treated with glucocorticoids or 7-irradiation. treatments thathave previously been shown to result in apoptosis (32-34, 70. 71).

Cleavage of pADPRp accompanied the apoptosis induced by thesetreatments (Fig. 9).

Effect of Protease Inhibitors on pADPRp Cleavage, DNA Fragmentation, and Cell Survival. In an attempt to characterize theprotease(s) involved in cleavage of pADPRp. HL-60 cells were incubated with etoposide for 1 h. washed, and incubated in drug-free

medium in the absence or presence of various protease inhibitors. Avariety of agents including phenylmethylsulfonyl fluoride, sodium

Puromycin1h 2 3 4 6

Oh 4 8 12 24 8 12 24

1

0.6-

116-

85-

B C-2-10

Fig. 6. Puromycin induces endonucleolytic DNA degradation (A) and cleavage ofpADPRp (B\. HL-60 cells were incubated with 100 ¿IMpuromycin for the indicated time.In separate experiments, this concentration of puromycin was found to inhibit (Õ5S|-methionine incorporation into trichloroacetic acid-precipitable material by 95<#.

1

0.6-

116- —¿�

85

B HL-60 K562

Fig. 7. Resistance of K562 chronic myelogenous leukemia cells to etoposide-inducedDNA fragmentation and pADPRp cleavage. HL-60 cells (lanes 1-5) or K562 cells (lanes6-10} were simultaneously incubated with 68 JJ.Metoposide for 0 h (lanes I and 6). 4 h

(lanes 2 and 7). 8 h (lanes 3 and 8), 12 h (lanes 4 and 9), or 24 h (lanes 5 and 10}.Duplicate samples were subjected to agarose gel electrophoresis (A) or SDS-PAGEfollowed by Western blotting with antibody C-2-10 (B). Similar results were obtainedwhen K562 cells were harvested after 48 h. Additional experiments indicated that theformation uf K562 colonies in soft agar was reduced to 10% of control values by a 1-hincubation in IO JÃŒMetoposide.

A TFT 5-FdUrd

Oh 12 24 48 Oh 12 24 48

B

OhN A

6N A

5-FdUrd

12 18NANA

24 48NANA

- - -116

-85

Fig. 8. Cleavage of pADPRp in human breast cancer cells undergoing apoptosis. In A,MDA-MB-468 cells were treated with IOO fiu trifluorothymidine (TFT) or 5-fluoro-2'-deoxyuridine (5-FdUrd) for 0—48h. Adherent and nonadherent cells were combined.Samples containing 50 jig of protein from each sample were blotted with antibodyC-2-IO. In ß.nonadherent (N) and adherent (A) MDA-MB-468 cells treated with IOO/IM5-FdUrd for 0-48 h were processed separately. Western blotting was performed as in A.

Although 50 ng of protein was loaded for each sample, the actual proportion of proteinin the nonadherent cells increased from 4.2<7rof the total (adherent + nonadherent) protein

at 0 h to 12.5% of the total at 48 h.

bisulfite. E-64, benzamidine. leupeptin, antipain, and phosphorami-

done had no effect on the cleavage of pADPRp (see Fig. 10 forexamples). In contrast, the chloromethyl ketones TLCK and tosyl-L-phenylalanine chloromethyl ketone as well as the sulfhydryl blockingreagents iodoacetamide, W-ethylmaleimide, and />-chloromercuriben-zenesulfonate all prevented cleavage of pADPRp to the Mr —¿�85.000

3981

CLEAVAGE OF POI.Y(ADP-RIBOSE) POI.YMKRASK

fragment (Fig. 10B, lanes 3-5, 8, and 9). Interestingly, each of theseagents prevented etoposide-induced internucleosomal DNA fragmentation (Fig. KM, lanes 3-5, 8, and 9) and the morphological changes

of apoptosis as well (Fig. IOC). Similar results were obtained inHL-60 cells and rat thymocytes after -y-irradiation (data not shown).

Despite these effects, these compounds did not prevent etoposide- orirradiation-induced cell death. On the contrary, cells treated with

iodoacetamide or TLCK (in the presence or absence of etoposide)uniformly excluded trypan blue for 2-3 h and then became permeable

to this dye. After 6 h of treatment, all of the cells treated withiodoacetamide and up to 80% of the cells treated with TLCK werepermeable to trypan blue. Thus, agents such as TLCK and iodoacetamide prevented the biochemical and morphological changes of apoptosis but did not prevent cell death.

DISCUSSION

In this present study, we have shown that proteolysis of pADPRp toan M, —¿�85,000fragment displaying DNA-independent enzyme activ

ity is an early event that accompanies apoptosis induced by a varietyof chemotherapeutic agents. The location of the proteolytic site on thepADPRp molecule and the biological significance of this observationare discussed below.

Cleavage of pADPRp. When HL-60 cells were treated with etoposide, the polypeptide recognized by monoclonal antibody C-2-10(Fig. 2C) showed a shift in mobility from M, 116,000 to -85,000.

Although the activation of transglutaminase has been observed inother cells undergoing apoptosis (reviewed in Ref. 81), the increasedmobility of pADPRp was not due to the formation of intramolecular¡sopeptide bonds. Blotting with an antiserum directed against thesecond zinc finger of pADPRp revealed that the epitopes recognizedby this serum were recovered in a second fragment of Mr —¿�25,000

(Fig. 2D). This result strongly suggests that proteolytic cleavage ofpADPRp rather than the formation of intramolecular isopeptide bondsoccurs during apoptosis.

This apoptosis-associated proteolytic cleavage of pADPRp is

readily distinguished from the fragmentation of pADPRp that occursduring the course of normal protein turnover. During normal turnover, pADPRp is cleaved toward its carboxyl terminus to yield anenzymatically inactive M, 80,000 fragment containing the DNA-binding and automodification domains (82). In contrast, apoptosis-associated cleavage of pADPRp removes the amino-terminal domainto yield an M, —¿�85,000fragment that retains basal catalytic activitybut lacks the DNA-binding domain and cannot be stimulated by

nicked DNA (Fig. 3).The identity of the protease that is responsible for the cleavage of

pADPRp is currently unknown. Bruno et al. (83) reported that chlo-

romethyl ketones inhibited DNA fragmentation in a number of experimental systems in which apoptosis was induced. Our experiments

Control + DEX + RT

Oh 1 23468 1 234612346 8

1 2 345678 9 10 11 12 13 14 15 16 17 18

116-

85-

C-2-10

Fig. 9. Cleavage of pADPRp in rat thymocytes. Nonirradiated (lanes 1-12) or irradiated (lanes ¡3-lti) thymocytes were incubated in the absence (lanes 1-7 and 13-18) orpresence (lanes 8-12) of 1 /¿Mdexamethasone for the indicated time and then prepared forWestern blotting with antibody C-2-10. Under the conditions of this assay, >95% of the

untreated and treated cells excluded trypan blue during the first 8 h.

23-

Fig. 10. Effect of protease inhibitors on DNA degradation (A), cleavage of pADPRp(B), and cell morphology (C). In A and B, after a 1-h treatment with 6K |XMetoposide.HL-60 cells were incubated for 3 h in the absence of protease inhibitors (lane 2) or in thepresence of 10 mM iodoacetamide (lAAmide. lane .Õ).10 rmi yV-ethylmaleimide (NEM,lane V), 1 mM /i-chloromercuribenzenesulfonate (pCMBS. lane 5). 1 mM phenylmethyl-sulfonyl fluoride (PMSF, lane 6). 1 mvi TLCK (lane cV),0.2 HIMtosyl-i.-phenylalaninechloromethyl ketone (TPCK, lane 9), 2 /xg/ml phosphoramidone (phosphor, lane 10), or5 fig/ml anlipain (lane II). Untreated cells were examined in lanes 1 and 7. C, Cellmorphology as determined by phase contrast (left) and fluorescence microscopy (righi)after staining with bisbenzimide. 1,1', control HL-60 cells. 2,2', cells incubated for l hwith 68 ^iMetoposide followed by 5 h in fresh medium lacking protease inhibitors. 3,3',

cells incubated for l h with 68 JXMetoposide followed by 5 h in fresh medium containing0.5 mM TLCK.

confirm and extend those observations. The studies presented in Fig.10 suggest that pADPRp cleavage in HL-60 cells can be inhibited byirreversible sulfhydryl-blocking reagents as well as chloromethyl ketones but not by serine esterase inhibitors. The sulfhydryl-dependentcathepsins are inhibited by this spectrum of inhibitors (84—86).Interestingly, this class of proteases is also inhibited by Zn2+ (86, 87), a

cation that has been observed to prevent apoptosis in a variety ofsettings (88-90).'' Although these observations are all consistent with

the view that the protease involved in pADPRp cleavage might be asulfhydryl-dependent cathepsin, a number of additional considerations

'' Subsequent experiments have revealed that cleavage of pADPRp in etoposide-trealedHL-60 cells is also prevented by treatment of the cells with 1 mM ZnCl, (S. H. Kaufmann,

unpublished observations).

3982

CLEAVAGE OF POLY(ADP-RIBOSE) POLYMERASE

argue against this conclusion. Intracytoplasmic release of a sulfhydr-yl-dependent cathepsin would be expected to cause degradation of

many cellular polypeptides (85, 91). The observation that most cellular polypeptides remain intact during apoptosis (Fig. 2, B and E)appears to argue against the widespread accessibility of cathepsins B,H, or L to intracellular polypeptides. Likewise, the inability of leu-peptin or E-64, two other inhibitors of sulfhydryl-dependent cathepsins (84-86, 91), to inhibit pADPRp cleavage also argues against thepossibility that the enzyme involved is a sulfhydryl-dependent cathep

sin, although the inability of these inhibitors to penetrate into cells ina timely fashion might also explain these results. Further studies aretherefore required to identify the protease(s) involved in pADPRpcleavage.

Biological Consequences of pADPRp Cleavage. Previous resultsfrom a number of laboratories have shown that the internucleosomaldegradation of DNA in cells undergoing apoptosis is accompanied bydepletion of cellular NAD stores (see "Introduction"). This NAD

depletion has been attributed to the consumption of NAD by pAD-

PRp, which is in turn thought to be activated by internucleosomalDNA breaks that appear during apoptosis. On the other hand, directmeasurement of enzyme activity suggested a decrease in pADPRpactivity in thymocytes undergoing irradiation-induced apoptosis (52).

These results have been difficult to reconcile with each other.The simultaneous monitoring of NAD levels and pADPRp cleavage

described in Fig. 4 suggests that both of the previous interpretationsare correct. When 3-AB was applied to HL-60 cells at a concentrationthat selectively inhibits pADPRp (56), the etoposide-induced declinein NAD levels was delayed (Fig. 4C, lanes 7-12). This result (see also

Refs. 30, 31, and 51) directly implicates pADPRp in the consumptionof NAD that ordinarily occurs early during the course of apoptosis(Fig. 4C, lanes 1-4). Furthermore, when cells were incubated in theabsence of 3-AB, NAD levels began to decline prior to the earliestdetectable cleavage of pADPRp (Fig. 4, B and C, lanes l^t). This

observation suggests that the intact pADPRp molecule rather than theMT ~85,000 fragment is responsible for the initial consumption of

NAD. By the time that nucleosomal DNA fragments appear, however,levels of the M, 116,000 polymerase are diminishing and the M,—¿�85,000fragment is being formed (Fig. 4, lanes 4-6). Although thisM, —¿�85,000fragment might play a role in the continuing consumption

of NAD late in the course of apoptosis (Fig. 4C, lanes 5 and 6), thefragment appears to have much lower catalytic activity in the presenceof nicked DNA than does the intact polymerase (Fig. 35). The formation of this less active fragment (see also Fig. 9, lanes 15-18)

appears to provide an explanation for the decreasing pADPRp catalytic activity that has been previously reported during radiation-in

duced apoptosis (46, 52).Although it is tempting to speculate that the proteolytic cleavage of

pADPRp serves a feedback function by decreasing the activity ofpADPRp after it has been stimulated by DNA strand breaks, the datado not appear to support this model. In particular, treatment with 3-AB

delays NAD consumption (presumably by inhibiting pADPRp) butdoes not delay DNA fragmentation and pADPRp cleavage in theetoposide-treated HL-60 cells (Fig. 4, lanes 10-12). This result sug

gests that activation of pADPRp activity is not required for proteolyticcleavage of pADPRp. If the downregulation can occur without thepreceding upregulation, it is difficult to view this as a feedback mechanism. Instead, the cleavage of pADPRp appears to represent one ofseveral proteolytic events that commonly occur during apoptosis (seebelow).

Implications for Programmed Cell Death. Studies in a numberof laboratories are directed at elucidating the common features ofphysiological cell death (see "Introduction"). Because of its almost

ubiquitous activation during apoptosis (reviewed in Refs. 5, 26, and

27), much current effort is focused on the identification and characterization of the "apoptotic endonuclease." Other frequently observed

features of the apoptotic process such as the induction of the TRPM-2gene (92) or the requirement for new protein synthesis (70-74) havebeen shown to be absent from certain cell types [absence of TRPM-2

induction (14, 93); apparent absence of requirement for protein synthesis (58, 75-79)]. On the other hand, the proteolytic process de

scribed in the present work appears to be widespread.There is ample precedent for the suggestion that proteases might

play a role in apoptosis. Experimental results in the 1960s and 1970srevealed that cathepsin activity increased in tissues undergoing apoptosis (94, 95). Increased protein turnover rates were subsequentlydemonstrated in isolated thymocytes undergoing apoptosis (96). Morerecent results suggested that quantitatively abundant nuclear proteinssuch as pADPRp (58, 59) and lamin B (58, 59, 97) were proteolyti-

cally cleaved during apoptosis in the limited number of experimentalsystems examined. The present results extend these earlier studies bydemonstrating that specific cleavage of the M, 116,000 pADPRppolypeptide to an M, —¿�85,000fragment accompanies endonucleolytic

DNA degradation in a variety of model systems and appears to be ahallmark of chemotherapy-induced apoptosis.

Since treatments that inhibit this proteolytic cleavage also inhibitthe internucleosomal DNA degradation (Fig. 10/4; see also Ref. 83)and the morphological changes associated with physiological celldeath (Fig. IOC), the possibility that limited proteolysis is an important controlling factor in initiating apoptosis needs to be considered. Inthis context, the recent suggestion that the apoptotic endonuclease isa preexisting cellular enzyme which decreases in molecular mass ascells undergo apoptosis (34) raises the interesting possibility that aproteolytic process similar to the one described in the present paperplays a role in activating the apoptotic endonuclease. Further studiesare required to assess this possibility.

ACKNOWLEDGMENTS

We gratefully acknowledge the technical assistance of Tim Soos and LisaPrichard, editorial assistance of Ann Larocca, and advice and encouragementof Drs. N. Berger, G. de Murcia, K. Kohn, J. Shaper, and M. Charron. The kindgift of anti-F2 antiserum by G. de Murcia is greatly appreciated.

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