(CANCER RESEARCH 49, 5077-5082. September 15. 1989]

Arrest of Replication Forks by Drug-stabilized Topoisomerase I-DNA CleavableComplexes as a Mechanism of Cell Killing by Camptothecin1

Yaw-Huei Hsiang, Michelle G. Lihou,2 and Leroy F. Liu

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT

Camptothecin, which induces an unusual type of DNA damage bytrapping cellular topoisomerase I on chromosomal DNA in the form ofdrug-enzyme-DNA cleavable complexes, inhibits DNA synthesis andspecifically kills S-phase cells. ( '»treatmentof LI210 cells with aphidi-

colin, which is an inhibitor of replicative DNA polymerases, completelyabolished Camptothecin cytotoxicity, suggesting the involvement of DNAreplication in Camptothecin cytotoxicity. In order to study the role ofDNA replication in drug action, a cell-free SV40 DNA replication systemwas used in the present study. Camptothecin inhibited SV40 DNAreplication in this cell-free system only in the presence of topoisomeraseI. Addition of excess purified calf thymus DNA topoisomerase I to thisextract system in the presence of Camptothecin resulted in severe inhibition of SV40 DNA replication and the accumulation of linearizedreplication products, which contained covalenti}- bound DNA topoisom

erase I. We propose that the collision between moving replication forksand camptothecin-stabilized topoisomerase I-DNA cleavable complexesresults in fork arrest and possibly fork breakage, which are lethal toproliferating cells.

INTRODUCTION

Camptothecin, a plant alkaloid isolated from Camptothecaaccanii nata of the Nyssaceae family, is a potent antitumor drugwith a broad spectrum of antitumor activity (1-3). Brief phaseI clinical trials in the early 70s, however, failed because ofexcessive toxicity (4, 5). Renewed interest in Camptothecin as apotential clinical antitumor drug have come from the recentidentification of its molecular target and the elucidation of itsmechanism of action (6).

Earlier studies in cultured mammalian cells have shown thatCamptothecin inhibits both DNA and RNA synthesis, andinduces reversible fragmentation of chromosomal DNA (7-13).While inhibition of RNA synthesis is rapidly reversible upondrug removal, inhibition of DNA synthesis is only partiallyreversible, a phenomenon which may be related to the S-phase-specific cytotoxicity of Camptothecin (14, 15). The cellulartarget of Camptothecin has been suggested from recent studiesusing purified mammalian DNA topoisomerase I (6). In thepresence of Camptothecin, purified mammalian DNA topoisomerase I can induce extensive DNA damage in the form ofenzyme-linked DNA breaks (6). Studies of topoisomerase I-linked DNA breaks have led to the proposal that Camptothecininterferes with the breakage-reunion reaction of topoisomeraseI by trapping an abortive reaction intermediate, the cleavablecomplex (6). Exposure of this cleavable complex to a strongprotein dénaturant,such as SDS ' or alkali, results in breakage

of the phosphodiester bond and the covalent linking of a topoisomerase I molecule to the 3' phosphoryl end of the brokenDNA strand (6). The drug-stabilized, enzyme-DNA cleavable

Received 12/15/88; revised 3/27/89. 5/26/89; accepted 6/15/89.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.

1This work was supported by NIH Grants CA-39962 and CA-40884.1Present address: Queensland Institute of Medical Research, Herstin, Queens

land. 4004, Australia.3The abbreviations used are: SDS, sodium dodecyl sulfate; DMSO, dimethyl

sulfoxide.

complex can be rapidly reversed to a noncleavable form by anumber of treatments, including a brief heating at 65°C(6, 16).

More recently, Hsiang and Liu (16) showed that camptothecin-induced fragmentation of chromosomal DNA is due to theformation of reversible topoisomerase I-DNA cleavable complexes in cells (16). More than 90% of the cellular topoisomerase I molecules were trapped covalently on chromosomalDNA in LI210 cells treated with 25 ¿IMCamptothecin (16).Studies of camptothecin-resistant human lymphoblastic leukemia cells and TOPI deletion yeast strains have provided furthersupport that topoisomerase I is the cytotoxic target of camp-tothecin (17, 18).

Based on the proposed mechanism of action for camptothe-cin, the cellular level of topoisomerase I is predicted to be animportant parameter for drug cytotoxicity. A higher cellulartopoisomerase I level predicts greater Camptothecin cytotoxicity. However, unlike DNA topoisomerase II, which is abundantonly in proliferating cells, DNA topoisomerase I is abundantin both proliferating and quiescent cells (19, 20). Furthermore,the level of topoisomerase I appears relatively constant betweenG,, S, G2, and M phases of the cell cycle (19). The S-phasespecificity of Camptothecin, therefore, cannot be explainedsolely by topoisomerase I levels (14, 15). In order to understandthe cell-killing mechanism of Camptothecin, we have studiedthe effect of DNA replication on Camptothecin cytotoxicity.Our present results suggest that the S-phase-specific cytotoxicity of Camptothecin may be due to the interaction betweenmoving replication forks and drug-stabilized topoisomerase I-DNA cleavable complexes.

MATERIALS AND METHODS

Materials. DNA topoisomerase I from calf thymus glands was purified as described previously (21). Plasmid pUC.HSO (ori*) and pUC.8-4 (ori~) DNAs (22) were purified by phenol deproteinization of cleared

lysates followed by CsCl/ethidium isopyknic centrifugation and gelfiltration on an A-50m column. SV40 large T-antigen purified fromrecombinant adenovirus R284-infected CV-1 monkey cells by inuminoaffinity column was a kind gift from Dr. Thomas Kelly (JohnsHopkins Medical School). Camptothecin sodium salt (NSC 100880)and Camptothecin lactone (NSC 94600) were obtained from the DrugSynthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute. Camptothecin was dissolved either in water(sodium form) or in DMSO (lactone form), and was stored in aliquotsat —¿�20°C.Aphidicolin (3 HIM)(Sigma Chemical Co.) was dissolved in

DMSO.Potassium-SDS Coprecipitation Assay. Potassium-SDS coprecipita-

tion assay for protein-DNA complexes was done as described previously(23).

Clonogenic Assays. Mouse lymphoblastic leukemic (LI210) cellswere grown in Fisher's medium supplemented with 10% heat-inacti

vated fetal bovine serum, 100 units/ml of penicillin, 100 Mg/ml ofstreptomycin, and 2 m%i glutamine. Cytotoxicity was measured by amodification of the clonal assay of Chu and Fisher (24). Briefly,logarithmically growing L1210 cells (2 x 105/ml) were exposed to

sodium Camptothecin (1 //\t) in the presence or absence of aphidicolin(0, 0.75, 1.5, 7.5, and 15 ¿IM,respectively). After a 1-h drug treatment,cells were washed twice with ice-cold phosphate-buffered saline prior

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REPLICATION FORK ARREST AND CELL DEATH INDUCED BY CAMPTOTHECIN

to plating in a semisolid agar medium (0.27% Bacto-Agar) in 35-mmplastic Petri dishes (200 or 500 cells/ml/dish). Colonies of greater than40 cells were scored by using a dissecting microscope (x40) on day 7.Results were expressed as mean survival (percentage of control) ±SE(four independent experiments). Control values were determined foreach aphidicolin concentration with appropriate dilutions of DMSO.DMSO (1%) at the lowest aphidicolin concentration reduced survivalto approximately 80% of untreated cultures. Aphidicolin did not furtherreduce survival, except at the highest dose (15 p\t), where survival was77% of the DMSO-treated control.

DNA Replication Assays in a Cell-free System. Replication of SV40origin containing plasmid DNAs in a HeLa cytosolic extract supplemented with purified SV40 large T-antigen was performed as described(25). Reactions (20 /jl each) containing 10 ß\extract, 0.5 ng of SV40 Tantigen and 50 ng of plasmid DNA in a standard reaction mixture (40mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid-KOH, pH 7.5,8 mM MgCh, 0.5 mM dithiothreitol, 100 n\\ concentrations each.ofdATP, dTTP, dGTP, 40 mM phosphocreatine, 1 mg/ml creatine phos-phokinase, 0.025% Nonidet P-40, 200 /,M CTP/UTP/GTP, 100 MMdATP/dTTP/dGTP, 25 ^M dCTP, 3 mM ATP, and 25 ^M [«-"P]dCTPwith a specific activity of 12 x 10' dpm/pmol) were incubated at 37°C

for 1 h. Reactions were terminated by the addition of SDS (finalconcentration, 1%) and proteinase K (final concentration, 1 mg/ml),and incubation was continued at 37°Cfor 90 min. Unless otherwise

indicated, DNA in the reaction mixtures was precipitated with ethanoland dissolved in 25 ^1 of 10 mM Tris, pH 8.0, 10 mM EDTA, and 5%sucrose. Electrophoresis was done in a 1% agarose gel (0.089 M Tris-borate, 0.002 M EDTA buffer). Gel drying and autoradiography weredone as described (26).

RESULTS

Inhibition of DNA Replication by Aphidicolin AbolishesCamptothecin Cytotoxicity. In order to study the effect of DNAreplication on camptothecin cytotoxicity, L1210 cells weretreated with sodium camptothecin (1 ÕÕMfor 1 h) in the presenceof increasing concentrations of aphidicolin, an inhibitor ofDNA polymerases a and 5 (27, 28). As shown in Fig. \A, inthe absence of aphidicolin, camptothecin treatment reduced cellsurvival to 55%. Increasing the camptothecin concentrationdoes not lead to further cell killing (data not shown), consistentwith previous reports that camptothecin selectively kills S-phase cells (14, 15), which represent approximately 45% of thetotal asynchronously growing cells (29). In the presence ofaphidicolin, survival of camptothecin-treated cells increased ina dose-dependent manner and 100% survival was achieved atconcentrations of aphidicolin above 7.5 ^M, where greater than95% of DNA synthesis was inhibited. At the same aphidicolinconcentration, camptothecin-induced DNA damage in L1210cells as monitored by the potassium-SDS coprecipitation assaywas unchanged (Fig. IB).

Inhibition of SV40 DNA Replication in the Cell-free SystemRequires Simultaneous Presence of Both Purified DNA Topoi-somerase I and Camptothecin. The effect of aphidicolin oncamptothecin cytotoxicity could indicate a requirement of DNAreplication in drug action. One possible explanation is thatcamptothecin cytotoxicity may require interaction betweenmoving replication forks and the camptothecin-induced topoi-somerase I-DNA cleavable complexes. This hypothesis wastested in a cell-free replication system which supports SV40 T-antigen- and origin-dependent DNA replication. Replication ofthe pUC.HSO DNA, which contains the origin sequence (nu-cleotides 5171-128), in this extract system resulted in the formation of both monomeric closed circular DNA products anda high-molecular-weight form DNA product (Fig. 2, Lane A).Addition of either camptothecin or purified calf thymus DNAtopoisomerase I alone to this extract system had little effect on

V)

2-5 5 7.5 IO 12.5

Aphidicolin Concentration

15

B

10000

8000

6000

4000

g **»

a.a

c3eU

25 125 825

Camptothecin Concentration (n M)

Fig. I. Aphidicolin protects LI210cells from camptothecin cytoxicity./4, drugtreatment and clonogenic assays were done as described in "Materials andMethods." B, camptothecin-induced protein-linked DNA breaks in the presence

and absence of aphidicolin. Camptothecin lactone was added immediately afterthe addition of aphidicolin. After 30 min of drug incubation, cells were processedfor potassium-SDS coprecipitation assays as described in "Materials and Methods."

SV40 DNA replication (Fig. 2, Lanes B-E and K-N). However,addition of both camptothecin and purified DNA topoisomerase I together to this extract system resulted in severe inhibitionof DNA replication and the formation of altered replicationproducts (Fig. 2, Lanes F-I and O-R). Since topoisomerase Icatalytic activity is not essential for DNA replication in thisextract system due to the presence of topoisomerase II (30), therequirement of both camptothecin and topoisomerase I forreplication inhibition suggests that the formation of drug-stabilized topoisomerase I-DNA cleavable complexes is likely tobe the cause of replication inhibition. The level of the endogenous topoisomerase I in this HeLa cytosolic extract is apparently too low to form sufficient cleavable complexes in thepresence of camptothecin (30).

Accumulation of Altered Replication Products in Presence ofTopoisomerase I and Camptothecin. In the absence of exogenoustopoisomerase I and camptothecin, replication of SV40 origin-containing plasmid DNA pUC.HSO resulted in the formationof monomeric closed circular daughter molecules (migrating asa group of topoisomer bands) and a diffuse slow-migratingDNA species (see Fig. 2, Lane A). However, in the presence ofcamptothecin and exogenous topoisomerase I, replication products migrating to the positions expected for linearizedpUC.HSO (form III) and nicked pUC.HSO (form II) accumulated (Fig. 2, Lanes F-I and O-R). In addition, a diffuse band,possibly representing replicative intermediates, also accumu-

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REPLICATION FORK ARREST AND CELL DEATH INDUCED BY CAMPTOTHECIN

ABCDEFGHI JKLMNOPQR

Fig. 2. Inhibition of SV40 DNA replicationand accumulation of linearized replication products require the simultaneous presence of bothpurified DNA topoisomerase I and camptothecin.SV40 DNA replication in vitro was done as described in "Materials and Methods." Lanes A andJ, control, DNA only. Lanes B-E. 20. 4, 0.8, and0.16 ng of purified calf thymus DNA topoisomerase I, respectively. Lanes F-l, same as Lanes B-E,respectively, except that 1 mm camptothecin sodium salt was added. Lanes K-N, 1, 0.2, 0.04, and0.008 MIMcamptothecin sodium salt, respectively.Lanes O-R, same as Lanes K-N, respectively, except that 20 ng of purified calf thymus topoisomerase I was added. pUC.HSO (II) denotes nickedpUC.HSO DNA; pUC.HSO (III) denotes linearized pUC.HSO DNA.

fi? Ititi pUC.HSO (II)pUC.HSO (III)

lated and had an electrophoretic mobility slower than that ofnicked pUC.HSO (form II) (Fig. 2, Lanes F and O-R).

Camptothecin has been shown to produce primarily protein-linked single-strand DNA breaks (6, 16). The formation of thelinearized replication product in the presence of camptothecinand topoisomerase I was therefore unexpected. In order tounderstand its formation, the linearized replication product wasfurther characterized. Like the nicked replication product (formII), the linearized replication product (form III) was shown tobe protein linked by its sensitivity to phenol extraction (Fig. 3).If, prior to proteinase K treatment, the replication reactionproducts were extracted with phenol, all replication productsformed in the presence of topoisomerase I and camptothecinexcept a small population of form I pUC.HSO DNA wereretained in the phenol-water interphase (Fig. 3, Lanes D, F, //,

and J).To test whether the formation of the linearized replication

product in the presence of camptothecin and topoisomerase Iis due to DNA replication, plasmid pUC.8-4 DNA, which isidentical to pUC.HSO except for a 4-base pair deletion withinthe SV40 origin of replication (22), was used in the extractsystem containing camptothecin and topoisomerase I. As revealed by Southern blot analysis, the amounts of nicked pUC.8-

4 and pUC.HSO DNA generated were about the same (Fig.4B\ compare Lanes D and //). However, more linearizedpUC.HSO DNA (form III) was generated than linearizedpUC.8-4 DNA (Fig. 4Ä;compare Lanes D and H). This linearized pUC.HSO DNA migrated at the same position as thelinearized replication product of pUC.HSO DNA (Fig. 4, LanesD). Furthermore, the band of the linearized pUC.HSO DNAshowed a slight downward smear in both gels, suggesting thepossibility that it may contain a heterogeneous population withsome linearized pUC.HSO DNA being less than the unit size.These results together suggest that the formation of linearizedpUC.HSO DNA is the result of DNA replication in the presence of camptothecin-induced topoisomerase I-DNA cleavablecomplexes.

The formation of linearized pUC.HSO DNA in the extractmay reflect the interaction between replication forks and camptothecin-induced topoisomerase I-DNA cleavable complexes.Such an interaction may lead to alterations in some propertiesof the cleavable complexes. Since the linearized pUC.HSOreplication product was covalently linked to protein, possiblydue to the presence of drug-induced topoisomerase I-DNAcleavable complexes, we tested the reversibility of this protein-DNA interaction with a number of treatments (a brief heat

AB CDEFGH I J

Fig. 3. Covalent association of topoisomerase I with plasmidDNA replicated in cell-free extracts supplemented with purifiedtopoisomerase I and camptothecin. Replication in HeLa cytosolicextract supplemented with purified SV40 T antigen was performedas described in "Materials and Methods." For each reaction (20

>i\). 20 ng of purified calf thymus DNA topoisomerase I wasadded. Lanes A, C, E, G, and /. 0, 1, 0.2, 0.04. and 0.008 mMsodium camptothecin was added, respectively. Following replication, reaction mixtures were treated with proteinase K (l mg/ml)at 37'C for 90 min prior to phenol extraction. Lanes B, D, F, H,

and J, same as Lanes A, C, E, G, and /, respectively, except thatproteinase K treatment was omitted. pUC.HSO (I) denotes super-coiled pUC.HSO DNA.

pUC.HSO(II)pUC.HSO(III)

pUC.HSO(I)5079

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REPLICATION FORK ARREST AND CELL DEATH INDUCED BY CAMPTOTHECIN

Fig. 4. Formation of linearized replication products in presence of camptothecin and topoisomerase Iis replication dependent. I DNA replication in HeLacytosolic extracts was done as described in "Materialsand Methods." ¡MnesA-D, pUC.HSO (ori*); LanesE-F, pUC.8-4 (ori~). Lanes A and £,control, DNA

only. Lanes B and /. 20 ng of purified calf thymusDNA topoisomerase I were added. Lanes C and G,0.008 HIMsodium camptothecin was added. Lanes Dand //. both lopoisomerase I and sodium camptothecinwere added. Elcctrophoresis was carried out by usinga 1% agarose gel in 0.089 M Tris-borate, 0.002 MEDTA buffer containing 0.5 jjg/ml ethidium bromide.B, all samples arc the same as in I except thai |»' I*|

dCTP was replaced with unlabeled dCTP. Followingelectrophoresis, the agarose gel was dried and probedwith nick-translated pUC.HSO DNA by in situ Southern hybridization (31).

AABCDE FGH

BABCD E FGH

ABCD

II (dimer)

III (dimer)

II (monomer)I (dimer)III (monomer)

I (monomer)Fig. S. Replication produces covalent topoisomerase I-DNA complexes with

altered reversibility. DNA replication in HeLa cytosolic extracts was performedas described in "Materials and Methods." For each reaction (20 i<l). 20 ng of

purified calf thymus DNA topoisomerase I was added. Lane A, no drug. LanesB-D. 0.008 HIMsodium camptothecin was added. To test the reversibility of thelopoisomerase 1-DNA complexes, reactions were briefly heated to 65"C for Omin

(Lane B), 1 min (Lane C), and 3 min (Lane D) prior to termination with SDSand proteinase K. Elcctrophoresis was carried out by using a I % agarose gel in0.089 M Tris-borate. 0.002 M EDTA buffer containing 0.5 Mg/ml ethidiumbromide.

treatment at 65°C,drug removal, addition of excess DNA, and

challenge with 0.5 M NaCl) known to reverse cleavable complexes (6, 16). Fig. 5 shows the result of such an experiment:the incubated replication reaction mixture was briefly heated to65°Cprior to termination of the reaction with SDS and pro

teinase K. While the majority of nicked pUC.HSO DNA replication product (form II) was converted to its closed circularform (form I), the amount of linearized pUC.HSO DNA replication product remained unchanged (Fig. 5; compare Lanes B

11(dimer)

lll(dinwr)

II(monomer)I (dinner)

-III (monomer)

I (monomer)

111*211*

and C). Similar results were obtained when the reaction waschallenged with 0.5 M NaCl or excess pBR322 DNA (20-fold)(data not shown). These results suggest that the topoisomeraseI-linked linearized pUC.HSO DNA replication product may beproduced by an altered cleavable complex, which apparentlyhas lost its characteristic reversibility. The loss of reversibilityof the cleavable complex is probably due to the collision of thereplication fork with the cleavable complex, as explained in"Discussion."

DISCUSSION

As proposed previously, the drug-induced topoisomerase I-DNA cleavable complex is central to the action of camptothecinin cells (6, 16). This hypothesis has gained strong support froma number of recent studies (17, 18). However, it is still unclearhow the formation of reversible topoisomerase I-DNA cleavablecomplexes leads to rapid tumor cell killing. Studies on the cell-killing mechanism of topoisomerase II poisons have suggestedthat the formation of topoisomerase II-DNA cleavable complexes in cells is necessary but not sufficient for cell killing (32,33). Interaction of certain cellular processes with the drug-stabilized cleavable complex has been suggested to be essentialfor the expression of drug cytotoxicity (32, 33). In view of thesimilarity between the cleavable complexes induced by topoisomerase I and II poisons, it seems plausible that a cellularfunction(s) may interact with camptothecin-induced topoisomerase I-DNA cleavable complexes to express the lethal effect ofcamptothecin. The S-phase-specific cytotoxicity of camptothecin suggests that DNA replication is the critical cellular function (14, 15). Our present result, that inhibition of DNA replication by the DNA polymerase inhibitor, aphidicolin, abolishescamptothecin cytotoxicity, lends further support for such aproposition. The complete protective effect of aphidicolin appears specific for camptothecin as only partial protection byaphidicolin can be observed for the topoisomerase II poisonVM-26 (teniposide).4 This result may indicate that, in addition

to DNA replication, other cellular processes may also interactwith topoisomerase II-DNA cleavable complexes to express thelethal effect of topoisomerase II poisons.

Our results from studies using the cell-free SV40 DNAreplication system can best be explained by the model shown

' Unpublished results.

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IIIIIIIIMIIIIIH

'I IMllIl

Replication Fork Arrest And Cell Death

Fig. 6. A schematic illustrating replication fork arrest by a drug-abortedtopoisomerase I-DNA cleavable complex. In this model, the camptothecin-trapped topoisomerase I-DNA cleavable complex is viewed as a bulky DNA lesionwhich arrests the replication fork by blocking the movement of replicationmachinery. This blockage also alters the physical state of the cleavable complexand possibly leads to fork breakage at the complex site. At low levels of cleavablecomplexes, when only one replication fork is arrested, continued replication bythe other unimpeded fork on the same plasmid DNA leads to the formation oflinearized replication products. The irreversible replication arrest and fork breakage may be the cause of camptothecin S-phase-specific cytotoxicity.

in Fig. 6. The camptothecin-induced topoisomerase I-DNAcleavable complex arrests the replication fork, presumably because of the covalent nature of the complex. Inhibition of RNAtranscription in an in vitro T7 RNA polymerase transcriptionsystem has also been shown to require the simultaneous presence of both camptothecin and purified mammalian DNAtopoisomerase I." These results suggest that the drug-induced

cleavable complexes may physically block the movement ofboth RNA and DNA polymerases in cells, and thereby inhibitRNA and DNA synthesis. The reversible inhibition of RNAsynthesis by camptothecin may be explained by the reversibilityof the drug-induced topoisomerase I-DNA cleavable complexes.However, DNA synthesis inhibition by camptothecin has beenshown to be only partially reversible. This paradox may beexplained by our observation that the arrest of the replicationforks is also accompanied by the formation of what could bealtered topoisomerase I-DNA cleavable complexes which canno longer be reversed by a number of treatments known toreverse cleavable complexes. The mechanism of altered cleavable complex formation in this in vitro replication system in thepresence of camptothecin and topoisomerase I is unclear. Onepossibility is that the collision between a replication fork and acleavable complex can lead to alteration of the cleavable complex, causing the formation of protein-linked DNA breaks inthe absence of a protein dénaturant.Alternatively, the arrest ofthe replication forks by covalent protein-DNA complexes mayalter the local DNA structure near the replication forks. For

example, the presence of single-stranded DNA regions near thearrested replication forks may lead to the formation of complexes between topoisomerase I and single-stranded DNA. Suchcomplexes have been shown previously to undergo spontaneousDNA cleavage in the absence of a strong protein dénaturantand therefore cannot be readily reversed to the uncleaved stateby treatments known to reverse cleavable complexes betweentopoisomerase I and double-stranded DNA (34). In either case,the collision between the fork and the complex can lead tobreakage of at least one arm (leading or lagging strand) of thereplication fork. The topoisomerase I linked, linearized replication products in the presence of low level of topoisomeraseI-DNA cleavable complexes may be produced by the arrest ofa single replication fork while the other fork on the samecircular template proceeds unimpeded to completion.

The effect of camptothecin on SV40 DNA replication hasbeen studied in SV40 virus-infected cells (35). Camptothecintreatment of these SV40-infected cells predominantly inducessigma-shaped replication intermediates rather than the linearized replication products produced in a cell-free system. Onepossible explanation for this apparent discrepancy may be dueto replication on a eliminatili template versus a naked DNAtemplate. SV40 DNA in minichromosome form has a stablenucleosome near the region of replication termination whichmay impede the movement of replication forks in both directions (36). Within cells, the blockage (and hence breakage) of asingle replication fork by the camptothecin-topoisomerase I-DNA cleavable complex results in the accumulation of sigma-shaped replication products rather than the linearized replication products produced in the cell-free system.

The effect of aphidicolin on camptothecin-induced aberrantreplication products has also been studied in SV40 virus-infected cells (35). Aphidicolin-camptothecin cotreatment doesnot affect the production of sigma-shaped replication products.This result argues against a direct collision between a movingDNA polymerase complex with camptothecin-induced cleavable complexes. However, fork movement may continue in theabsence of functional polymerases. It has been shown previouslythat aphidicolin inhibits SV40 DNA replication but does notarrest the replication forks (37). Apparently, the T antigenhelicase can continue its helix-unwinding function while movement of polymerases is inhibited. The uncoupling between helixunwinding (and hence fork movement) and chain polymerization has also been demonstrated in a purified system usingSV40 T antigen and single-strand DNA binding protein (38,39). The lack of a protective effect by aphidicolin againstcamptothecin-induced fork breakage in the SV40 system (35)is in contrast to the protective effect of aphidicolin againstcamptothecin cytotoxicity in LI210 cells. One possible explanation may be a tighter coupling between the cellular replicativehelicase (in contrast to T-antigen helicase in SV40 DNA replication) and chain polymerization in 1.1210 cells. Inhibition ofchain polymerization by aphidicolin may effectively block forkmovement and therefore prevent the lethal collision betweenadvancing replication forks and camptothecin-induced topoisomerase I-DNA cleavable complexes. The generation of linearized replication products in the presence of camptothecinhas also been observed in SV40 virus-infected monkey cells(40). However, the linearized replication products can be chasedinto replicated circular daughter molecules (40). It seems possible that the replication forks may be broken at the site of thecleavable complexes but the broken forks are still held in closeproximity by the replication complexes. Further experimentsare necessary to establish the detailed interaction between the

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replication forks and the cleavable complexes.In summary, arrest of the replication forks by camptothecin-

induced topoisomerase I-DNA cleavable complexes is likely tobe the primary cause of camptothecin cytotoxicity. Detailedanalysis of the mechanism of fork arrest should provide crucialinformation concerning the mechanism of tumor cell killing bytopoisomerase I poisons, and unveil the general principlesinvolved in cancer chemotherapy.

ACKNOWLEDGMENTS

We wish to thank Dr. C. Bret Jessee for technical assistance and Dr.Erasmus Schneider and Kawai Lau for critical reading of the manuscript.

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1989;49:5077-5082. Cancer Res   Yaw-Huei Hsiang, Michelle G. Lihou and Leroy F. Liu  CamptothecinI-DNA Cleavable Complexes as a Mechanism of Cell Killing by Arrest of Replication Forks by Drug-stabilized Topoisomerase

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