(CANCER RESEARCH 49, 5870-5878. November 1, 1989]

Induction of Endonucleolytic DNA Cleavage in Human Acute MyelogenousLeukemia Cells by Etoposide, Camptothecin, and Other CytotoxicAnticancer Drugs: A Cautionary Note1

Scott H. Kaufmann2

Oncology Center, Johns Hopkins Hospital, and Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine,Baltimore, Maryland 21205

ABSTRACT

Treatment of human III -60 or KG1A leukemia cells with the topoi-somerase II inhibitor etoposide resulted in extensive DNA degradation.When DNA integrity was analyzed by agarose gel electrophoresis, anucleosomal ladder became evident 1.5-2 h after addition of etoposide tocells, increased in intensity over 6 h, and persisted at 24 h. Six h afteraddition of the drug, 94 ±4% of the cells excluded trypan blue eventhough as much as 90% of the DNA had been degraded to oligosomalfragments. Exposure of cells to 10 «ig/ml(17 ¡IM)etoposide for as littleas 45 min was sufficient to induce this DNA damage 4 h later. Preincu-bation with dinitrophenol abolished the effect of etoposide, suggestingthat an energy-requiring step occurred prior to or during the endonucleo-lytic cleavage. In contrast, the effect of etoposide was not prevented bypreincubation of 111.-6(1cells with the RNA synthesis inhibitor 5,6-dichloro-l-0-ribofuranosylbenzimidazole or the protein synthesis inhibitors cycloheximide or puromycin. On the contrary, high concentrationsof 5,6-dichloro-l-/3-ribofuranosylbenzimidazole, cycloheximide, or puromycin by themselves induced the same endonucleolytic cleavage, as dida variety of diverse cytotoxic agents, including camptothecin (0.1 n\\).colcemid (0.1 fig/ml), c/j-platinum (20 MM),methotrexate (1 MM),and I/3-D-arabinofuranos) Icytosine (3 UM). These results suggest that endonucleolytic DNA damage by a preexisting cellular enzyme occurs soonafter treatment of 111.-60 cells with any of a variety of cytotoxic agents.The observation that a variety of nuclear proteins [including poly(ADP-ribose) polymerase, lamin B, topoisomerase I, topoisomerase II, andhistone HI) are degraded concomitant with the DNA fragmentation callsinto question the selectivity of the degradative process for DNA. Theimplications of these results for (a) current theories which focus uponendonucleolytic damage of DNA as a critical early event during celldeath, and (b) use of topoisomerase-directed drugs to map topoisomerase-binding sites in active chromatin are discussed.

INTRODUCTION

The epipodophyllotoxins etoposide3 and teniposide are po

tent and widely used antineoplastic agents. A variety of differentexperiments suggest that the first step in the mechanism ofaction of these agents involves the stabilization of a covalentadduci between the nuclear enzyme topoisomerase II and thegenomic DNA (reviewed in Refs. 1-3; see also Refs. 4-6).Protein-linked DNA single- and double-strand breaks resultfrom this interaction (6, 7).

Several experiments have demonstrated a correlation betweenepipodophyllotoxin-induced DNA strand breaks and cytotox-

Reccived 3/1/89; revised 5/15/89; accepted 7/13/89.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported in part by grants from the American Cancer Society

(Maryland Division). The Andrew Mellon Foundation, and by Grant CA 06973from the N1H.

2To whom requests for reprints should be addressed, at Oncology 1-127. JohnsHopkins Hospital. 600 N. Wolfe St., Baltimore. MD 21205.

' The following abbreviations are used: etoposide (VP-16). 4'-demethylepipo-dophyllotoxin-9-(4.6-O-ethylidene-/J-D-glucopyranoside): teniposide (VM-26).4'-demcthylepipodophyllotoxin-9-(4.6-O-thionylidine-/i-D-glucopyranoside); cis-platinum, ci'5-diamminedichloroplatinum(ll); PMSF, phenylmethylsulfonyl fluoride; DRFB. 5,6-dichloro-l-0-ribofuranosylbenzimidazole;SDS, sodium dodecyl-sulfate: CHO. Chinese hamster ovary.

icity (e.g., Refs. 6 and 7). Other observations, however, call intoquestion the direct relationship between topoisomerase Il-me-diated strand breaks and cell death. First of all, the correlationbetween strand break frequency and cytotoxicity disappearswhen one compares topoisomerase II-directed agents with differing structures (2, 8). Second, analysis of topoisomerase II-mediated strand breaks and cytotoxicity after treatment ofsynchronized HeLa cells with 4'-(9-acridinylamino)meth-

anesulfon-m-anisidide, another topoisomerase II-directedagent, has also failed to show a direct correlation between strandbreak frequency and cytotoxicity (9). Instead, DNA strandbreaks formed during S phase appear to be particularly cytotoxic. Third, etoposide-induced strand breaks are rapidly re-sealed after removal of the drug (6, 10), but the reseating ofthese strand breaks does not prevent cell death. Conversely,treatment of murine erythroleukemia cells with dinitrophenoldiminishes etoposide-induced cytotoxicity but does not preventformation of topoisomerase II-DNA adducts (11) and consequent strand breaks. All of these observations suggest that oneor more steps subsequent to the formation of protein-linkedDNA strand breaks might be important in the mechanism ofepipodophyllotoxin-induced cell death.

Recent results from several laboratories suggest that drug-induced endonuclease activity might be an important step whichfollows the inhibition of topoisomerase II by various agents.Villeponteau et al. (12) reported that novobiocin, a topoisomerase II inhibitor which does not stabilize DNA-protein crosslinks, induces endonucleolytic cleavage in embryonic chickenerythrocytes. When erythrocytes at a specific stage of embryonicdevelopment were treated with this agent, a nucleosomal pattern of DNA degradation (characteristic of the action of anendonuclease on the internucleosomal linker regions of DNAin situ) was rapidly produced. More recently, Jaxel et al. (13)reported similar endonucleolytic DNA fragmentation aftertreatment of mitogen-stimulated lymphocytes with teniposideor with camptothecin, an agent which stabilizes the covalentadduct between topoisomerase I and DNA (14-17). It was notreported whether this effect was specific for lymphocytes or forthese particular agents. Likewise, it was not clear whether theDNA degradation was a cause or a consequence of the cytotoxicity of these drugs.

The drug-induced endonucleolytic DNA damage describedabove is reminiscent of DNA fragmentation observed aftertreatment of lymphocytes with cytotoxic concentrations of glu-cocorticoids in vitro (18-22) and in vivo (23, 24). In lymphoidcells, the degradation of DNA is specific for glucocorticoids (asopposed to other steroids, e.g., Ref. 24) and is prevented bysimultaneously incubating the cells with inhibitors of proteinor RNA synthesis (Refs. 20, 24, 25; for contrary data, see Refs.19 and 26). This latter result suggests that synthesis of a newprotein is required for endonuclease action and cell death.While definitive identification of the pertinent gene producthas not been established, these data have been interpreted as

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indicating that glucocorticoids induce the synthesis of an en-

donuclease (reviewed in Refs. 27 and 28), that this inducedendonuclease extensively damages the cellular DNA (27, 28),and that this extensive DNA damage in turn sets into motiona series of events (24, 26, 27), including activation of enzymessuch as poly(ADP-ribose) polymerase which deplete the cell ofNAD* and other compounds containing high-energy phosphate

bonds (22, 29, 30), thus leading to the cessation of cellularmetabolism.

In the present study, the effect of epipodophyllotoxins orcamptothecin on DNA integrity in human leukemia cell lineshas been examined. Within 2 h after treatment of log phaseHL-60 or KG1A cells with cytotoxic doses of these agents,extensive endonucleolytic DNA damage is evident. Exposuresas brief as 20-30 min are sufficient to induce this damage 4 hlater. In contrast to the glucocorticoid-induced DNA fragmentation in lymphoid cells, the epipodophyllotoxin-induced DNAdamage in acute myelogenous leukemia cell lines is unaffectedby simultaneous incubation with cycloheximide at concentrations which inhibit protein synthesis by 90%. In fact, treatmentwith higher concentrations of cycloheximide or with high concentrations of DRFB, an RNA synthesis inhibitor, by themselves induce DNA fragmentation in HL-60 or KG1A cells.Similar endonucleolytic damage also appears at varying timesafter treatment with cw-platinum, methotrexate, l -ß-D-arabi-nofuranosylcytosine, or colcemid. These observations suggestthat the appearance of endonucleolytic DNA damage in acutemyelogenous leukemia cell lines does not require new proteinsynthesis and is not specific for inhibitors of topoisomer-ases, but instead reflects the cytotoxicity of any of a variety ofcompounds.

MATERIALS AND METHODS

Materials. Etoposide and teniposide were kind gifts of Bristol-MyersPharmaceutical Co. (Syracuse, NY). Camptothecin, podophyllotoxin,methotrexate, l-/3-D-arabinofuranosylcytosine, colcemid, rà-platinum,puromycin, cycloheximide, DRFB, ATP, and iV-2-hydroxyethylpiper-azine-N'-2-ethanesulfonic acid were from Sigma Chemical Co. (St.Louis, MO). Agarose and \Hind\\\ molecular-weight markers werefrom Bethesda Research Laboratories (Rockville, MD). Tissue culturemedia were from M. A. Bioproducts (Walkersville, MD). Fetal bovineserum was from GIBCO (Grand Island, NY). [3H]Uridine was fromAmersham (Arlington Heights, IL). íra/ií-[35S]Methioninewas from

ICN (Irvine, CA). All other chemical reagents were obtained as previously described (31-33).

Buffers. TE9S contained 500 HIMTris-HCl (pH 9.0 at 21°C),2 mM

EDTA, 10 mM NaCl, and 1% (w/v) SDS. TAE consisted of 40 mMTris-acetate and l mM EDTA. Hypotonie buffer 1 contained 10 mMTris-HCl (pH 7.4 at 4°C),10 mM NaCl, 3 mM MgSO4, 0.1 mM EDTA,

100 units/ml Trasylol, 0.5 mM dithiothreitol, 1 mM 2-mercaptoethanol,and 1 mM PMSF. Hypotonie buffer 2 consisted of 5 mM potassiumphosphate (pH 7.0 at 21°C),2 mM MgSO4, 0.1 mM EDTA, 0.5 mM

dithiothreitol, 1 mM 2-mercaptoethanol, and 1 mM PMSF (34). Buffer3 consisted of 50 mM Tris-HCl (pH 7.5 at 4°C),100 mM KC1, 10 mM

MgSO4, 0.5 mM dithiothreitol, and 0.5 mM EDTA (35). STM buffercontained 250 mM sucrose, 50 mM Tris-HCl (pH 7.4 at 4°C),and 5

mM MgSO4. Alkylation buffer contained 6 M guanidine hydrochloride,250 mM Tris-HCl (pH 8.5 at 21°C),10 mM EDTA, 150 mM 2-

mercaptoethanol, and 1 mM PMSF. PMSF was added immediatelyprior to use to aliquots of the indicated buffers from a 100 mM stockin anhydrous 2-propanol.

Incubation with Drugs. Human HL-60 or KG1A myelogenous leukemia cells (kindly provided by Drs. W. S. May and C. Civin, JohnsHopkins Oncology Center, respectively) were grown at 37°Cin RPMI

1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum,100 units/ml penicillin G, 100 Mg/ml streptomycin, and 2 HIMgluta-

mine (complete medium) in an atmosphere containing 5% (v/v) CO2.Cells were harvested while in log phase (0.6-1.0 x IO6 cells/ml) andpurified by Ficoll-Hypaque sedimentation (33). Human mononuclearcells and granulocytes were purified from EDTA-treated blood bysedimentation on Ficoll-Hypaque step gradients.

Cells were sedimented at 400 x g and resuspended at a concentrationof 1-2 x IO6 cells/ml in fresh 1-ml aliquots of complete mediumcontaining 10 mM /V-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

acid (pH 7.4). Various concentrations of etoposide, teniposide, camptothecin, 1-0-D-arabinofuranosylcytosine, or methotrexate were addedin 1-jil aliquots of dimethyl sulfoxide (final concentration, 0.1%, v/v).All other agents were added from stocks prepared in complete medium.After varying lengths of incubation at 37°C,cells were sedimented at

3300 x g for 1 min and lysed in 35-75 ^1 TE9S buffer containing 1

mg/ml proteinase K.Purification and Analysis of DNA. Samples in TE9S buffer containing

proteinase K were incubated at 48°Cfor 24-48 h, extracted twice with

phenol/chloroform and once with chloroform as described by Goelz etal. (36), treated for 1-3 h at 21°Cwith 300 Mg/ml boiled bovine

pancreatic RNase A, and loaded onto 1.2% (w/v) agarose horizontalslab gels containing 10 pg/m\ ethid ium bromide. DNA from 5 x IO5

cells was loaded into each lane. Electrophoresis was performed in TAEbuffer for 12-15 h at 1.5 V/cm. Gels were photographed under UVlight with Polaroid type 665 film.

Incubation of Isolated Nuclei. HL-60 cell nuclei were isolated byincubating cells at 4°Cfor 20 min in hypotonie buffer 1 or 2, disrupting

the cells with a precooled tight-fitting Dounce homogenizer, and sedi-menting the nuclei at 800 x g. Alternatively, nuclei were prepared byincubating cells at 4°Cwith STM buffer containing 1% (w/v) Nonidet

P-40 and sedimenting the nuclei. For each of these methods, the releaseof cytoplasm-free nuclei was monitored by phase contrast microscopy.Isolated nuclei were incubated at 37°Cin buffer 3 [a buffer which

permits topoisomerase II-mediated formation of cleavable complexes(4, 35)] in the presence of etoposide (40 Mg/ml) and various concentrations of ATP (0, 1, or 2.5 mM). Alternatively, nuclei were incubated at37°Cin STM buffer in the presence of various concentrations of CaCl2

(0, 1, or 2.5 mM), ATP (0, 1, or 2.5 mM), and etoposide (0 or 40 Mg/ml). Addition of ice-cold ethanol to a final concentration of 80% (v/v)stopped the incubations and precipitated the DNA. After sedimentation, DNA was purified and analyzed as described above.

Incorporation of |35S|Methionine and (3H]Uridine into Macromole-

cules. To assess the effect of cycloheximide or DRFB on protein orRNA synthesis over the time course of the experiments depicted in Fig.5, duplicate aliquots of HL-60 cells (1 x 106/ml in compiete medium)were incubated for l h at 37°Cin the presence of the indicatedconcentration of cycloheximide or DRFB. [35S]Methionine (1 j/Ci) or[3H]uridine (1 >iCi) was then added to the cycloheximide- or DRFB-

treated cells, respectively, and the incubation was continued for anadditional 3 h. Samples were sedimented at 3300 x g for 1 min andwashed once with 1 ml ice-cold tissue culture medium. Incorporationinto protein and RNA, respectively, were determined by precipitatingthese macromolecules with ice-cold 10% (w/v) trichloroacetic acid,washing the pellets with acetone, drying the samples, solubilizing with0.1 M NaOH (30 min, 70°C),neutralizing the samples with an equal

volume of 0.1 M HC1, and subjecting the supernatants to scintillationcounting as previously described (33).

Cloning of Cells in Soft Agar. Etoposide was added to log phase HL-60 cells from freshly prepared 1000-fold concentrated stocks in dimethyl sulfoxide. After 1 h, cells were sedimented at 400 x g for 10min and washed once with complete medium. Cells were then resuspended in complete medium and diluted into the agar-containing medium described by Pike and Robinson (37). Aliquots (1 ml) were platedin Lux gridded 35-mm Petri dishes (Nunc, Naperville, IL) and incubated for 10-14 days at 37°Cin a humidified atmosphere containing

7.5% (v/v) CO2 and 92.5% (v/v) air before colonies were counted.Cloning of cells treated with an equivalent volume of dimethyl sulfoxide(no drug) revealed a cloning efficiency of 8-12% (range in 4 experiments).

Western Blotting. As previously described (31, 32), samples for SDS-polyacrylamide gel electrophoresis were reduced in alkylation buffer,

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alkylated with iodoacetamide, and dialyzed sequentially into 4 M ureaand 0.1% SDS prior to lyophilization. After solubilization in SDSsample buffer at 70°Cfor 20 min, samples were applied to 0.75-mm

slab gels consisting of a separating gel with a 5-15% (w/v) linearacrylamide gradient and a stacking gel containing 5% (w/v) acrylamide.After electrophoresis, gels were stained with Coomassie blue or trans-fered to nitrocellulose as previously described (38).

Rabbit polyclonal antisera against human topoisomerase I and IIwere from Dr. Leroy Liu (Johns Hopkins University School of Medicine, Baltimore, MD). Mouse monoclonal anti-histone HI was fromDr. James Sorace (Veterans Administration Hospital, Baltimore, MD).Mouse monoclonal anti-poly(ADP-ribose) polymerase was from Dr.Guy Poirier (Laval University, Quebec City, Quebec, Canada). Antibodies against lamínB and the A/, 67,000 nuclear matrix polypeptidewere raised by previously published techniques (39, 40). Western blotting was performed as previously described (38) by using these primaryantibodies and radioiodinated secondary antibodies. After autoradiog-raphy, some blots were erased and reprobed with additional antibodies(38).

RESULTS

Nucleosome-like DNA Cleavage Induced by Etoposide. Mostexperiments investigating the formation of epipodophyllotoxin-induced DNA-protein cross-links involve analysis of DNA bysensitive techniques such as alkaline elution after a 30-60 minincubation of cells or nuclei with the drug. To search for moredelayed effects of epipodophyllotoxins on tumor cell DNA,HL-60 human progranulocytic leukemia cells were incubatedwith etoposide for varying lengths of time up to 18 h before theDNA was analyzed by agarose gel electrophoresis (Fig. I A).This technique is not sensitive enough to detect the strandbreaks induced as a consequence of drug-induced stabilizationof topoisomerase-DNA adducts [a few strand breaks/IO7 base

pairs (6)]. Consequently, l h after addition of etoposide to thecells, the DNA from drug-treated cells (Fig. I A, Lane 5) wasindistinguishable from the DNA in control cells (Fig. IA, Lane3). In both samples, the DNA remained near the top of the gel.By 2 h after addition of 40 Mg/ml (68 /JM)etoposide to the HL-60 cells, a ladder of DNA fragments ranging from about 200base pairs upward in multiples of 170 base pairs became evidenton the gels (Fig. I A, Lane 6). These DNA fragments comigratedwith nucleosomal fragments of DNA prepared by treating isolated HL-60 cell nuclei with micrococcal nuclease (Fig. IA,Lane 2). Between 2 and 6 h after addition of the etoposide, theamount of high-molecular-weight DNA decreased and theamount of DNA fragments increased (Fig. IA, Lanes 6-9). Thisnucleosomal ladder remained evident at 18-24 h after additionof the drug (Fig. IA, Lane 10).

Preliminary experiments to assess the metabolic integrity ofthe cells utilized trypan blue exclusion assays. Six h after theaddition of 40 Mg/ml etoposide, 94 ±4% (n = 4) of the HL-60cells excluded trypan blue. Thus plasma membrane integritywas intact at a time when the genome had already sustainedextensive damage.

To better characterize the conditions required for inductionof the DNA cleavage, HL-60 cells were incubated with varyingconcentrations of etoposide (Fig. IB). Similar DNA cleavagewas evident within 4 h after the start of treatment with 10 Mg/ml (17 UM)etoposide (Fig. \B, Lane 4), a concentration whichkills 90-95% of the cells in a clonogenic assay.4 Treatment with

teniposide at concentrations as low as 1 ng/m\ (1.5 MM)likewiseresulted in detectable DNA cleavage 4 h after the start oftreatment (Fig. IB, Lane 12).

'S. H. Kaufmann, unpublished observations.

To assess whether continued exposure to etoposide was required for the appearance of DNA damage, HL-60 cells wereincubated with etoposide (40 /¿g/ml)for varying lengths of time,washed, and incubated without etoposide for the remainder of4 h (Fig. 1C). Exposure to 40 ng/m\ etoposide for 20-30 minwas sufficient to induce detectable DNA fragmentation at 4 h(Fig. 1C, Lanes 4 and 5). Additional experiments have shownthat exposure to 100 /¿g/mletoposide for 3 min or 10 ng/mlfor 45 min results in similar DNA damage at 4 h (data notshown).

The presence of the nuclear enzyme topoisomerase II appeared to be required for induction of the epipodophyllotoxin-induced endonucleolytic damage. Etoposide treatment of cellswhich lack topoisomerase II4 such as human granulocytes (Fig.

ID) or resting lymphocytes (not shown) did not result inendonucleolytic DNA damage. In contrast, etoposide-inducedDNA damage was observed in a number of cell lines containingtopoisomerase II including KG1A human leukemia cells (Fig.IE) and log-phase CHO cells (Fig. 2). In the case of the latter

cell line, it is important to note that the time lag between theaddition of etoposide and the appearance of DNA fragmentswas substantially longer than in the acute myelogenous leukemia cell lines. In additional experiments, DNA fragmentationfirst became evident in log-phase CHO cells 18 h after additionof etoposide to cultures.

Camptothecin Induces Similar Endonucleolytic DNA Damagein Human Leukemia Cell Lines. When HL-60 cells were treatedwith the topoisomerase I inhibitor camptothecin, a nucleosomalladder of DNA fragments again became evident by 2 h afteraddition of the drug (Fig. 3A, Lane 4). The amount of DNAfragments again increased and the amount of high-molecular-weight DNA decreased between 2 and 6 h after addition of thedrug (Fig. 3A, Lane 4-7). Treatment of HL-60 cells with 1 MMcamptothecin for periods as brief as 20-30 min was sufficientto produce DNA fragmentation 4 h later (Fig. 3B, Lanes 4 and5). Similar DNA fragmentation was observed in KG1A cells(Fig. 3C) but not in human granulocytes or resting lymphocytes(not shown). Concentrations of camptothecin as low as 0.1 MMwere sufficient to induce the DNA damage in susceptible cells(Fig. 3D, Lane 3).

Endonucleolytic DNA Damage Is Prevented by Treatment withDinitrophenol. To assess the possibility that an energy-requiringstep might be needed between treatment of the cells withtopoisomerase-directed agents and the appearance of DNAfragmentation, cells were incubated with the oxidative phos-phorylation uncoupler dinitrophenol for l h before addition ofetoposide or camptothecin. Preincubation with dinitrophenolcompletely prevented the endonucleolytic fragmentation induced by these drugs (Fig. 4, Lanes 6 and 7, respectively). Thecytotoxicity of etoposide or camptothecin as assessed by clonogenic assay was also diminished by this dinitrophenol treatment.4 Further experiments were undertaken to assess whetherthe energy-requiring step(s) suggested by these results mightinvolve RNA or protein synthesis (see below).

Endonucleolytic DNA Damage Is Not Prevented by Inhibitorsof Protein and RNA Synthesis. The DNA damage induced inHL-60 cells by epipodophyllotoxins or camptothecin was rem

iniscent of the DNA damage induced in thymocytes and inlymphoma cell lines after treatment with glucocorticoids (see"Introduction"). In these lymphoid cells, it has been suggested

that glucocorticoids enhance the expression of a gene encodingfor an endonuclease which digests the DNA (27, 28). To assessthe possibility that the etoposide-induced DNA damage wasthe result of enhanced expression of a gene encoding for an

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O 0.5 1 2 3 4 6 18h 0VP-16

40 20 10 4 2 1 0.4 40 4

VM-26

2 1 0.4 0.1

Fig. 1. DNA fragmentation as a consequence of epipodophyllotoxin treatment. A,time course of etoposide-induced DNA fragmentation in HL-60 cells. HL-60 cells weretreated with 40 /jg/ml (68 /JM)etoposide for 0,0.5, 1, 2, 3, 4, 6, or 18 h (Lane 3-10, respectively). DNA was isolated and subjected toagarose gel electrophoresis as described in"Materials and Methods." The migration of\Hind\\\ molecular-weight markers (Lane 1)and the DNA degradation pattern obtainedafter incubating isolated HL-60 nuclei withmicrococcal nuclease (Lane 2) are shown forcomparison. Ordinate, sizes of \Hind\\\ fragments in kilobase pairs. B, dose-response curveof epipodophyllotoxin-induced DNA degradation in HL-60 cells. HL-60 cells were incubated for 4 h with 40, 20, 10, 4, 2, 1, or 0.4¿ig/mletoposide (Lanes 2-8) or 40,4, 2, 1,0.4,or 0.1 Mg/ml teniposide (Lanes 9-14) beforeisolation of DNA for agarose gel electrophoresis. The DNA of cells incubated in the absence of added epipodophyllotoxin is shownfor comparison (Lane I). C effect of briefexposure of HL-60 cells to etoposide. HL-60cells were treated at 37°Cwith 40 ^g/ml eto

poside in complete medium for 0, 10, 20, 30,45, or 60 min (Lanes 2-7, respectively), sedi-mented. and incubated in drug-free mediumfor the remainder of 4 h. For comparison,DNA from cells incubated in the presence of40 ng/ml etoposide for the entire 4 h is shownin Lane 1. D, incubation of mature granulo-cytes with epipodophyllotoxin does not resultin DNA fragmentation. Ficoll-Hypaque-puri-fied granulocytes from human peripheralblood were incubated with 40 Mg/ml etoposidefor 0. 1. 3, or 6 h prior to isolation of DNA(Lanes 1-4). Also shown is DNA from granu-locyte incubated for 6 h in the absence ofetoposide (Lane 5). Etoposide did not induceDNA cleavage in these cells. Western blotting(not shown) revealed that mature granulocyteslack the topoisomerase II polypeptide. E, etoposide-induced DNA cleavage in M.I \ cells.KG IA cells were incubated with 40 fig/mletoposide for 0, 2, 3, 4, 6, 12, or 26 h (Lanes1-7, respectively) prior to isolation of DNAfor agarose gel electrophoresis.

0.56

Oh 2 4 11 25 36

0.56-

Fig. 2. Time course of DNA fragmentation in Chinese hamster ovary cells.CHO cells (50% confluent) in Dulbecco's modified Eagle's medium supplemented

with 10% (v/v) bovine serum and 2 mM glutamine were incubated with 40 >ig/mletoposide for 0, 2, 4, 7, 11, 25, or 36 h (Lanes 2-8, respectively). Adherent cellswere removed from their substratum by treatment with trypsin-EDTA, sedi-mented, and extracted as described in "Materials and Methods." Hindlll size

markers (ordinate) are shown for comparison (Lane I).

endonuclease, HL-60 cells were preincubated for l h withvarying concentrations of cycloheximide or puromycin (proteinsynthesis inhibitors) or DRFB, an RNA synthesis inhibitor,followed by the addition of etoposide for 3 h. One such experiment is shown in Fig. 5. Treatment with cycloheximide at <10fig/ml inhibited 35S incorporation into protein by as much as

90% but did not prevent the effect of etoposide (Fig. 5, Lanes7-9).5 Higher concentrations of cycloheximide (e.g., 50 Mg/ml)

inhibited protein synthesis by >95% and simultaneously induced DNA damage which was indistinguishable from thatinduced by etoposide (Fig. 5, Lane 6). Similar results wereobtained with puromycin (data not shown). Likewise, treatmentwith the RNA synthesis inhibitor DRFB at 1-3 ng/ml did notprevent the effect of etoposide (Fig. 5, Lanes 15 and 16);whereas higher concentrations of DRFB by itself induced DNAdamage similar to that induced by etoposide (Fig. 5, Lanes 13and 14). These results suggest that RNA and protein synthesis

' Similar results have been obtained by using brief exposures to low concentra

tions of etoposide (e.g., preincubation with 1 ng/ml cycloheximide for 1 h,followed by addition of 10 Mg/ml etoposide for 1 h, followed by a 3-h incubationwith 1 pg/ml cycloheximide in the absence of etoposide).

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O 0.5 1 10 20 30 45 60m + DNP

0.56

0.56

O 2 3 4 6 12 26h 100 10 4 1 0.3 OxlO"7M

Fig. 3. DNA fragmentation as a consequence of camptothecin treatment. A,time course of camptothecin-induced DNA fragmentation in HL-60 cells. HL-60cells were treated with l UMcamptothecin for 0, 0.5, 1, 2, 3, 4, 6, or 18 h (Lanes1-8, respectively). DNA was isolated and subjected to agarose gel electrophoresisas described in "Materials and Methods." Ordinales, migrations and sizes (inkilobase pairs) of \Hind\\\ molecular-weight markers. B, effect of brief exposureof HL-60 cells to camptothecin. HL-60 cells were treated at 37°Cwith 1 MMcamptothecin in complete medium for 0, 10, 20, 30, 45, or 60 min (Lanes 2-7,respectively), sedimented. and incubated in drug-free medium for the remainderof 4 h. For comparison, DNA from cells incubated in the presence of 1 MMcamptothecin for the entire 4 h is shown in Lane I. Nonadjacent wells from asingle gel were juxtaposed to prepare this panel. C, camptothecin-induced DNAcleavage in KG1A cells. KG1A cells were incubated with 1 CMcamptothecin for0, 2, 3, 4, 6, 12, or 26 h (Lanes 1-7, respectively) prior to isolation of DNA foragarose gel electrophoresis. D, dose-response curve of camptothecin-inducedDNA degradation in KG1A cells. KG1A cells were incubated for 4 h withcamptothecin at 10 MM(Lane 1), l MM(Lane 2), 0.4 ¿IM(Lane 3), 0.1 MM(Lane4), or 0.03 MM(Lane 5). Shown for comparison is the DNA from cells incubatedfor 4 h in the absence of camptothecin (Lane 6). DNA degradation in a nucleo-somal pattern was evident after 4 h of incubation with doses of camptothecin aslow as 0.1 MM(Lane 4). Similar results were obtained in HL-60 cells (data notshown).

are not required for expression of the etoposide-induced DNA

damage. Further, these results indicate that treatment of cellswith agents other than topoisomerase inhibitors will result insimilar endonucleolytic DNA damage.

Mechanistically Diverse Chemotherapeutic Agents Induce Endonucleolytic DNA Damage in Acute Myelogenous LeukemiaCells. The preceding results suggest that inhibitors of topoisomerase II (Fig. 1), inhibitors of topoiosmerase I (Fig. 3), inhibitorsof RNA synthesis (Fig. 5) and protein synthesis (Fig. 5), all are

0.6

Fig. 4. Dinitrophenol (DNP) prevents the endonucleolytic damage induced byetoposide or camptothecin. HL-60 cells were incubated at 37°Cfor l h in completemedium without (Lanes 2-4) or with (Lanes 5- 7) 10 mM dinitrophenol. Etoposide(40 Mg/ml. Lanes 3 and 6) or camptothecin ( 1 MM.Lanes 4 and 7) was added; andthe incubation was continued for 4 h. Cells were sedimented and DNA wasanalyzed as previously described. Nonadjacent wells from a single gel have beenjuxtaposed to compose this figure. \Hind\\\ markers are shown in Lane I forcomparison. The endonucleolytic damage induced by etoposide (Lane 3) orcamptothecin (Lane 4) was completely prevented by the presence of dinitrophenol(Lanes 6 and 7, respectively).

able to induce endonucleolytic DNA damage in acute mye-logenous leukemia cell lines. To assess whether other agentsmight also induce similar DNA fragmentation, HL-60 cellswere treated with methotrexate, 1-0-D-arabinofuranosylcyto-sine, c/s-platinum, or colcemid, four agents with diverse mechanisms of action. Endonucleolytic cleavage was evident aftertreatment with each of these agents (Fig. 6, Lanes 9-12,15,17,and 18). The time course of appearance of the DNA fragmentation, however, varied considerably. Extensive DNA fragmentation was evident within 6 h after addition of 1-0-D-arabino-furanosylcytosine to the cultures (Fig. 6, Lane 10). In contrast,DNA fragmentation did not become prominent until 24-48 hafter addition of methotrexate (Fig. 6, Lanes 8 and 9) orcolcemid (Fig. 6, Lane 17) to the cells.

DNA Fragmentation Is Not Due to Action of an EndogenousNuclear Protein. The preceding results suggest that a variety ofdifferent cytotoxic agents are all capable of inducing endonucleolytic DNA damage. The observation that inhibitors of RNAand protein synthesis did not prevent this drug-induced DNAdamage suggested that the endonuclease involved is an endogenous cellular protein. Endonucleases which might mediate thisdamage have been reported in the nuclei of a variety of cells(41-43). To assess the possibility that an endogenous nuclearprotein might mediate the etoposide- and camptothecin-induced DNA damage, nuclei isolated from untreated HL-60cells by three different methods were incubated at 37°Cfor upto 4 h under a variety of conditions (see "Materials and Methods"). DNA was then isolated and subjected to agarose gel

electrophoresis. Two such experiments are shown in Fig. 7. InFig. 1A, nuclei prepared from cells swollen in hypotonie bufferwere incubated with 40 ßg/m\etoposide for up to 4 h at 37°C

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VP-16

CHD DRFB

1234 10 11 12 13 14 15 16 17 18

Fig. 5. Preincubation with inhibitors of RNA or protein synthesis does notprevent etoposide-induced DNA fragmentation. Log phase HL-60 cells wereincubated for l h with cycloheximide (CHD) at 0 (Lanes 1 and 2), l ¿ig/ml(Lanes3 and 7), 3 ng/ml (Lanes 4 and 8), 10 fig/ml (Lanes 5 and 9), or 50 Mg/ml (Lanes6 and 10), or with DRFB at 1 fig/ml (Lanes II and 15). 3 ng/ml (¿an«/2 and16), 10 /ig/ml (Lanes 13 and 77), or 50 /ig/ml (Lanes 14 and /A). Etoposide wasthen added to a final concentration of 40 jjg/ml (Lanes 2, 7-10, 15-18). After 3h of additional incubation, cells were lysed and DNA was analyzed by agarose gelelectrophoresis. Low concentrations of cycloheximide (Lanes 7-9) did not preventthe effect of etoposide even though these concentrations inhibited [35S]methionine

incorporation into protein by up to 90%. A higher concentration of cycloheximide(which inhibited protein synthesis by >95%) by itself induced endonucleolyticDNA cleavage (Lane 6) which was indistinguishable from that induced by a 3-hincubation with etoposide alone (I.tine 2). Likewise, low concentrations of DRFBdid not prevent the effect of etoposide (Lanes 15 and 16), whereas higherconcentrations of DRFB (Lanes 13 and 14) which inhibited [3H]uridine incorpo

ration into RNA by up to 85% also induced endonucleolytic DNA damage.Similar results have been obtained by using KG1A cells rather than HL-60 cells.Likewise, similar results have been obtained by using l /IMcamptothecin in placeof etoposide.

cleave DNA (4, 35). A nucleosomal ladder was not producedunder these conditions. In subsequent experiments addition ofATP (1 or 2.5 HIM) or Ca2+ (a required cofactor for the calcium-, magnesium-dependent class of nucleases) in the absenceor presence of etoposide (40 Mg/ml) also failed to generate anucleosomal ladder from the DNA of nuclei prepared by ho-mogenization of cells under hypotonie conditions (data notshown). Likewise, when nuclei were prepared by disruptingcells in nonionic detergent, subsequent incubation did not resultin a nucleosomal ladder (Fig. IB, Lane 1). On the other hand,incubation of these same nuclei with the detergent-containingcytoplasmic extract did result in a nucleosomal ladder (Fig. IB,Lane 2). This latter result confirms that untreated HL-60 cellscontain an endonuclease capable of generating the nucleosomalDNA degradation pattern described in previous experiments.In addition, this experiment suggests that the endonuclease isnormally limited to an extranuclear compartment of the cell.

Extensive Protein Degradation Accompanies Etoposide-induced DNA Fragmentation. The results presented in Fig. IBraised the possibility that the drug-induced endonucleolyticdamage described in Figs. 1-6 might reflect the action of anendogenous cellular hydrolase which is normally present in anextranuclear compartment of the cell, possibly in lysosomes. Ifleakage of hydrolases from an extranuclear compartment intothe nucleus were responsible for the drug-induced DNA degradation, one might also expect to observe extensive hydrolysisof nuclear proteins over a similar time course. To assess thispossibility, HL-60 cells were treated with etoposide for various

VP-16 lengths of time, solubili/ed under strongly denaturing conditions [6 M guanidine hydrochloride containing 1% (v/v) 2-mercaptoethanol], and subjected to SDS-polyacrylamide gelelectrophoresis, followed by Western blotting with antibodiesdirected against a variety of nuclear proteins. Results of onesuch experiment are shown in Fig. 8. One h after addition ofetoposide to the cells, all of the nuclear proteins were presentin amounts similar to those found in control cells (cf. Fig. 8,B-G, Lanes 1 and 5). By 2 h, however, poly(ADP-ribose)

polymerase, a M, 116,000 nuclear enzyme, had undergoneextensive degradation to a M, 85,000 fragment (Fig. SB, Lane6). At the same time, the level of lamin B, the major structuralprotein of the HL-60 nuclear envelope, had diminished by 75%

(cf. Fig. 8C, Lanes 1 and 6). By 4 h after addition of etoposide,the amounts of topoisomerase II, topoisomerase I, and histoneHI had also markedly diminished (cf. Fig. 8, D-F, Lanes 1 and8). These results suggest that extensive degradation of manynuclear proteins accompanies the drug-induced DNA degradation described above.

DISCUSSION

In the present study treatment of human acute myelogenousleukemia cell lines with achievable therapeutic levels of epipo-dophyllotoxins (44) has been shown to result in the delayedcleavage of tumor cell DNA to a nucleosome-like pattern.Exposure of the cells to drug for 20-30 min is sufficient toinduce extensive DNA degradation in acute myelogenous leukemia cell lines 4 h later (Fig. 1C). Current models stress thestabilization of covalent adducts between topoisomerase II andthe cellular DNA as an important early event in the cytotoxicmechanism of epipodophyllotoxins (1-6). The present data donot conflict with these models. The relative potencies of theepipodophyllotoxins in inducing the endonucleolytic cleavage(teniposide > etoposide s> podophyllotoxin) parallels the potencies of these compounds in stabilizing topoisomerase II-DNA adducts (5, 6). More importantly, cells which lack topoisomerase II such as granulocytes do not display epipodophyl-lotoxin-induced DNA fragmentation despite the presence of avariety of hydrolases which could degrade their DNA (Fig. ID).These data are consistent with the view that topoisomerase IImust be present if etoposide is to initiate the events which resultin DNA fragmentation.

On the other hand, several considerations suggest that stabilization of the covalent complex between topoisomerase IIand the cellular DNA (with the concomitant formation of DNAsingle- and double-strand breaks) is not by itself sufficient toaccount for the DNA fragmentation observed in the presentstudy. First of all, the frequency of double-strand breaks observed in this study (every 200-2000 base pairs) is orders ofmagnitude higher than the frequency of double-strand breaks(about 1 break per 106or 107base pairs) formed when etoposidestabilizes the topoisomerase II-DNA adducts (6). Second, formation of topoisomerase II-mediated strand breaks requires thecontinued presence of etoposide and is readily reversed uponthe removal of the drug (6,10), whereas the DNA fragmentationdescribed in the present study does not require the continuedpresence of etoposide (Fig. 1C) and is irreversible. Third, thetopoisomerase II-mediated DNA strand breaks are proteinlinked (1-7), whereas the double-strand breaks described in thisstudy are not.4 Fourth, the formation of topoisomerase II-DNA

complexes occurs when isolated nuclei are incubated with topoisomerase H-directed agents (2, 7); but the fragmentationobserved in the present study is not observed when nuclei are

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Fig. 6. Structurally diverse chemothera-peutic agents induce endonucleolytic DNAdamage in HL-60 cells. Ordinate, \Hind\l\size markers. HL-60 cells in complete mediumwas incubated for 6, 24. or 48 h in the absenceof chemotherapeutic agents (Lanes 2-4), for 6h in the presence of 40 Mg/ml etoposide (VP)(Lane 5). for 6, 24. or 48 h in the presence ofl MMmethotrexate (MTX) (Lanes 7-9). for 6,24, or 48 h in the presence of 3 MM 1-0-D-arabinofuranosylcytosine (ARA-C) (Lanes 10-12), or for 6, 24. or 48 h in the presence of 1Mg/ml colcemid (COL) (Lanes 16-18). Alternatively, cells in complete medium were incubated for I h with 20 MMc/j-platinum (PLAT),washed, and incubated for 0, 5. or 47 h morein complete medium without drug (Lanes 13-15, respectively.). \Hind\\\ molecular-weightmarkers are shown in Lane 1 for comparison.Nonadjacent wells from a single gel have beenjuxtaposed to compose this figure. DNA degradation was not evident in control cells butwas similar in pattern and amount 6 h afteraddition of etoposide (Lane 5). 48 h afteraddition of methotrexate (Lane 9), 24 h afteraddition of l-/i-D-arabinofuranosylcytosine(Lane II), 48 h after treatment with m-plati-iinni (Lane 15), and 24 h after addition ofcolcemid to these cells (Lane 17).

VP

hr 6 24 48 6

MTX ARA-CH I I H

PLAT COLH t-

6 24 48 6 24 48 1 48 6 24 48

1 2 3 7 8 9 10

0.6

Fig. 7. Incubation of isolated nuclei with etoposide does not result in DNAfragmentation. Ordinate, \Hind\l\ size markers. A, nuclei were isolated at 4°Cfrom untreated HL-60 cells by incubating the cells for 20 min in hypotonie buffer1, rupturing the swollen cells with 10 strokes of a tight-fitting Dounce homoge-nizer, and sedimenting the nuclei at 800 x g for 10 min. After a single wash,nuclei were treated with 40 Mg/ml etoposide in buffer 3 at 37°Cfor 0, 1, 2, or 4h (Lanes 2-5. respectively). At the completion of the incubation. DNA wasethanol precipitated and prepared for electrophoresis as described in "Materialsand Methods." The DNA fragmentation pattern obtained by incubating an equalnumber of intact HL-60 cells with 40 Mg/ml etoposide for 4 h is shown in Lane1. Incubation of isolated nuclei with etoposide did not result in DNA fragmentation under these conditions (Lanes 2-5) or a variety of other conditions (seetext). B, nuclei were isolated from untreated HL-60 cells at 4°Cby incubating thecells for 5 min with STM buffer containing 1% (w/v) Nonidet P-40 and sedimenting the nuclei. Nuclei resuspended in fresh STM buffer was incubated for 3h at 37*C prior to ethanol precipitation and analysis of DNA (Lane 1). As acontrol, untreated HL-60 cells were treated with STM buffer containing 1%Nonidet P-40, but the nuclei were not isolated. Instead, the whole cell lysate wasincubated at 37'C for 3 h prior to addition of 80% (v/v) ethanol (Lane 2). The

fragmentation of DNA in a nucleosomal pattern in the whole cell lysate (Lane 2)confirms that untreated HL-60 cells contain an endonuclease which is capable ofgenerating the DNA damage observed in previous figures. Under ordinary' conditions this endonuclease appears to be absent from nuclei (A. Lane 5: B, Lane1) but can gain access to nuclei after removal of membrane barriers (B. Lane 2).

incubated with the drugs (Fig. 1A). Finally, the binding ofepipodophyllotoxins to topoisomerase II in intact cells andresultant formation of protein-linked DNA strand breaks israpid [<30 min (6, 10)], whereas the DNA cleavage observed inthis study does not appear until at least 90 min after additionof etoposide to HL-60 cells or 18 h after addition of etoposideto CHO cells (Fig. 1 and 2).4 This time delay between drug

addition and extensive DNA fragmentation suggests the possibility of one or more intervening metabolic steps rather than adirect action of topoisomerase II on the DNA.

The DNA fragmentation observed in this study is reminiscentof the nucleosomal DNA degradation observed in lymphoidcells after treatment with cytolytic doses of glucocorticoids (see"Introduction"). In this latter system, it has been proposed that

glucocorticoids enhance the expression of an endonucleasewhich is responsible for the DNA damage and subsequent celldeath (Refs. 27 and 28; for contrary view, see Refs. 19 and 26).In the present study, treatment with inhibitors of RNA synthesis [DRFB (Fig. 5)] or protein synthesis [cycloheximide (Fig.5); puromycin, data not shown] at concentrations which diminished RNA or protein synthesis by 85-90% did not inhibit theeffect of etoposide on HL-60 cell or KG1A cell DNA integrity.Higher concentrations of these same RNA and protein synthesis inhibitors induced endonucleolytic damage themselves whilesimultaneously inhibiting RNA or protein synthesis to an evengreater extent. These observations make it unlikely that theendonucleolytic fragmentation described in the present studyresults from transcription and translation of a new gene product. Likewise, the inability to demonstrate an endogenous nu-clease in HL-60 cell nuclei isolated by three separate techniques(Fig. 7; see "Materials and Methods") makes it unlikely that

the endonuclease is an endogenous nuclear protein. These observations lead to the speculation that a cytoplasmic endonuclease, possibly of lysosomal origin, becomes active within thenucleus as a late consequence of treatment of susceptible cellswith epipodophyllotoxins or camptothecin.

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1h 2 346

200

31

pADPRp

Bmm

Lamín B Dm

mTopo II •¿�••

Topo I

H1

p67

-

Fig. 8. Extensive degradation of many nuclear proteins accompanies etopo-side-induced DNA degradation. Ordinate, molecular weight in thousands. Logphase HL-60 cells at a concentration of 1 x 106/ml in complete medium wereincubated with 40 fig/ml etoposide for 0 h (Lanes 1-4), l h (Lane 5), 2 h (Lane6), 3 h (Lane 7), 4 h (Lane 8), or 6 h (Lane 9). At the completion of the incubationthe cells were sedimented at 400 x g for 10 min, washed once in serum-freemedium, and solubilized in buffered 6 M guanidine hydrochloride containing ISOHIM 2-mercaptoethanol, 10 HIM EDTA, and 1 HIM PMSF (alkylation buffer).After reduction, alkylation, dialysis, and lyophilization (32), samples containingprotein from 3 x IO5cells (Lanes 1, 5-9), 1.5 x 10' cells (Lane 2), 0.75 x 10*cells (Lane 3). or 0.3 x 10*cells (Lane 4) were subjected to SDS-polyacrylamidegel electrophoresis followed by staining with Coomassie blue (A ) or transfer tonitrocellulose and Western blotting with mouse monoclonal anti-poly(ADP-ribose) polymerase (pADPRp) (B), chicken anti-lamin B (LamínB) (C), rabbitanti-topoisomerase II (Topo II) (D), rabbit anti-topoisomerase I (Topo I) (E),mouse monoclonal anti-histone HI (HI) (F), or guinea pig anti-P67 (p67) (G),followed by appropriate radiolabeled secondary antibody. Blotting with the anti-Pô?antibody revealed that P67, a polypeptide which appears relatively resistantto proteolysis, is present in similar amounts in Lanes I and 5-9, thus confirmingthe appropriate loading of the gels (G). Similar results were obtained withantibodies to the major nucleolar protein B23 (not shown). In contrast, manyother nuclear polypeptides were extensively degraded during the course of thisexperiment. Between 1 and 2 h after addition of etoposide, >75% of the M,116,000 poly(ADP-ribose) polymerase disappeared from the gel (cf. B, Lanes 5and 6, upper band). Simultaneously, a Mr 85,000 degradation product of thispolypeptide appeared (I!, lower band) and was more slowly degraded (cf. B, Lanes6 and 9). Within 2 h of addition of etoposide, there was also a 75% decrease inthe amount of lamin B, a major structural protein of the nuclear envelope (cf. C,Lanes 5 and 6). Between 2 and 4 h after addition of etoposide. there was also a>50% decrease in the amounts of topoisomerase II, topoisomerase I, and histoneH1 which could be detected in these cells (cf. Lanes 6-8, D, E, and F, respectively).Thus the degradation of DNA (Fig. IA) is accompanied by a simultaneousdegradation of many different nuclear polypeptides.

Previous studies have left the impression that inhibitors oftopoisomerase II (12, 13) and/or topoisomerase I (13) mightbe selective in inducing the endonucleolytic DNA damage. Inthe present study it was observed that a wide variety of agentsare able to set into motion the metabolic events which result inDNA fragmentation (Figs. 5 and 6). Among these are agentswhich act directly on DNA (cis-platinum) and agents which arethought not to interact with DNA at all (methotrexate andcolcemid). The induction of DNA fragmentation by thesewidely divergent agents suggests that DNA fragmentation is alate event which reflects impending cytotoxicity even though itoccurs long before cells lose the ability to exclude trypan blue.The observation that many nuclear proteins are simultaneouslydegraded (Fig. 8) suggests that the hydrolytic process is notunique to DNA.

The results described in the present study have implicationsnot only for certain models which envision that endonucleasedamage plays a critical role in the cytotoxicity of selectedcompounds, but also for current practice in molecular biology.Recent studies from several laboratories suggest that topoisomerase II-binding sites in active genes can be identified by treatingcells with epipodophyllotoxins and assessing the effect on theintegrity of the gene under study (45, 46). A similar techniquehas been utilized to map topoisomerase I-binding sites (16, 47-49). The interpretation of data obtained by these topoisomer-ase-mapping techniques relies upon the assumption that theDNA strand breaks observed in the presence of epipodophyllotoxins or camptothecin have been formed by the action oftopoisomerase II or topoisomerase I, respectively, and not bythe action of another nuclease. Some of these studies, however,have involved treating intact cells with topoisomerase-directeddrugs for up to 6 h (46). In light of the demonstration thattreatment of certain cells with these compounds can set intomotion metabolic events which rapidly result in extensive DNAdamage by an endonuclease (Ref. 13; this study; see also Ref.12), care will be needed in applying this method of mappingtopoisomerase-binding sites. The results presented in the current study suggest that exposure times to the drugs should bestrictly limited during topoisomerase mapping (16, 47, 48). Inaddition, these results suggest that it might be preferable totreat isolated nuclei (rather than intact cells) with these agentsin order to map topoisomerase-binding sites within genes (49,50).

Finally, it is important to note that the DNA degradationobserved in this study is not unique to acute myelogenousleukemia cell lines (Fig. 2; see also Refs. 12, 13, and 51). Whatis unique to these leukemia cell lines is the rapidity and extentof DNA degradation which is observed after treatment withepipodophyllotoxins or camptothecin. These observations suggest that the leukemia cell lines might prove useful for analyzingthe poorly understood sequence of events which occur betweenthe stabilization of topoisomerase-DNA covalent complexesand death of target cells. Likewise, the current observationsraise the possibility that treatment of clinical samples of leu-kemic blasts with various chemotherapeutic agents in vitrofollowed by assessment of DNA integrity might provide arelatively rapid screening test for sensitivity or resistance ofpatients' blasts to drug-induced cell death. Both of these pos

sibilities are under current investigation.

ACKNOWLEDGMENTS

The technical assistance of Sharon McLaughlin and the secretarialassistance of Ann Larocca are gratefully acknowledged, as are the kind

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gifts of antibodies from Drs. Leroy Liu, Guy Poirier, and James Sorace.Encouragement and advice from Drs. O. Michael Colvin, Leroy Liu,Richard J. Jones, Joel Shaper, and Nancy Shaper were greatly appreciated.

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1989;49:5870-5878. Cancer Res   Scott H. Kaufmann  Other Cytotoxic Anticancer Drugs: A Cautionary NoteMyelogenous Leukemia Cells by Etoposide, Camptothecin, and Induction of Endonucleolytic DNA Cleavage in Human Acute

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