(CANCERRESEARCH57. 1654-1659.MayI. 19971

Advances in Brief

Down-Regulation of DNA Replication in Extracts of Camptothecintreated Cells:Activation of an S-phase Checkpoint?1

Ya Wang, Ange Ronel Perrault, and George Hiakis2

Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Abstract

Extracts prepared from camptothecin (CPT)-treated cells have a reduced ability to support SV4O DNA replication in vitro. This reductionderives mainly from a reduction in the frequency of initiation events

because DNA chain elongation remains practically unchanged. Mixing ofextract from nontreated cells with small amounts of extract of CPTtreated cells indicates that the reduction In DNA replication is due to the

synthesis/activation of a dominant inhibitor. The observed reduction in

DNA replication activity cannot be attributed to inactivation oflopo I, themolecular target of camptothecin, because levels and activity of thisprotein remaln unchanged In extracts of CPT-treated cells and addition ofpurified lope I does not restore replication activity. Although replicationprotein A (RP-A) is phosphorylated in CFI-treated cells, reduced replication may not be caused by RP-A inactivation, because neither loss ofphosphorylation nor the addition of recombinant RP-A restore replication

activity. We interpret these observations as biochemical evidence for the

activation of a checkpoint in S phase and discuss the ramifications of thisactivation on the mechanismof CPT-inducedcytotoxicity.

Introduction

CPT3 and its analogs are agents with a unique spectrum of antitumor activity mediated by a selective inhibition of eukaryotic DNAtopoisomerase I (Topo 1; for recent reviews, see Refs. 1—3).Thecytotoxicity of these compounds is predominantly exerted during S

phase and is associated with an inhibition of DNA replication. Thisinhibition is generally thought to be primarily the result of a passivecollision of the advancing replication fork with the CPT-Topo I-DNAcleavable complex (4—8).Such collisions are expected to cause aninhibition in the elongation steps of DNA replication (9) and to killcells by generating DNA dsbs (10, 11). However, not all effects ofCF-F can be explained by the collision model, and recently, evidencewas presented that the sensitivity of cells to CPT may also be determined by their ability to activate checkpoints in S and 02 (12—14).

Because CPT toxicity is at maximum during S phase (1—3),wehypothesized that if regulatory processes, such as those initiated inresponse to the activation of a checkpoint in S phase (15, 16), preventreplication on damaged/altered DNA template, they will operate astoxicity modifiers. Indeed, inhibition of DNA replication by aphidicohn reduces CPT toxicity (4, 9, 13). DNA replication inhibition as aresult of the activation of regulatory processes is expected to have asimilar outcome, with the significant difference that the protective

Received 117/97; accepted 3/20/97.The costs of publication of this article were defrayed in pan by the payment of page

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

I This work was supported by National Cancer Institute Grant IRO1 # CA56706,

awarded by NIH, United States Department of Health and Human Services and by aUNCF-Merck Postdoctoral Research Fellowship Award.

2 To whom requests for reprints should be addressed, at Department of Radiation

Oncology, Thompson Building, Room Bl3, Thomas Jefferson University, Philadelphia,PA 19107.Phone:(215)955-6473;Fax:(215)955-2052;E-mail:Iliakisl@jeflin.tju.edu.

3 The abbreviations used are: CPT, Camptothecin; dsb, double-stranded break; TAg,large tumor antigen; ssDNA, single-stranded DNA; RP-A, replication protein A; PFGE,pulsed-field gel electrophoresis; Topo, topoisomerase; DNA-PK, DNA-dependent proteinkinase.

mechanism will be under cellular control. Information on the molecular determinants of this regulation may offer new ways of intervention for drug sensitization. Therefore, we sought biochemical cvidence for the operation of regulatory processes inhibiting DNAreplication in human cells exposed to CPT. We examined the abilityof extracts prepared from CPT-treated cells to support DNA replication using the SV4O based in vitro replication assay (17—19).In thisassay, replication of plasmids carrying the SV4O origin of DNA

replication is achieved with extracts prepared from human cells. Allproteins required for replication, except for the SV4O TAg, are ofcellular origin. The assay allows the evaluation of extracts preparedfrom CPT-treated cells for the presence of regulatory factors, as wellas for the inactivation of essential replication factors. Because the

plasmid DNA substrate is never exposed to CPT, complications

otherwise arising when damaged DNA is used as substrate (as with

experiments carried out in vivo) are eliminated. We have previouslyvalidated this assay in experiments designed to characterize the regulation of DNA replication in cells exposed to ionizing radiation (20,21).

Materials and Methods

Cell Culture and Drug Treatment. HeLa cells were grown in spinnerflasks seeded at 0.5—1X 10―cells/ml at volumes that ranged from 0.5 to 10liters for 3—4days at 37°Cin Joklik-modified MEM (S-MEM) supplementedwith 5% iron-supplemented calf serum (Sigma Chemical Co., St. Louis, MO).CVF sodium salt (NCS100800, obtained from the National Cancer Institute)

was added to mid-logarithmic phase cultures (4—6X l0@cells/ml) at 37°Cfor3 h. Cells were processed for extract preparation immediately after completionof treatment.

Cell Extract Preparation and in VitroAssay of DNA Replication. Themethods used for extract and TAg preparation, as well for the assembly of thein vitro reactions, have been described previously (20, 21). Extracts (S-lOO)

and TAg were stored in small aliquots at —70°C.The plasmid DNA,pSVO1AEP,carrying the minimal origin of SV4ODNA replication, was usedas template in the replication reactions (kindly provided by Dr. J. Hurwitz,Memorial Sloan-Kettering Cancer Center, New York, NY). The reactionmixture was incubated at 37°Cfor 1 h. Reactions were terminated by adjustingthe mixture to 20 mu EDTA. Activity present in acid insoluble material wasdetermined as described (19). Replication products were analyzed by agarosegel electrophoresis.

DNA Synthesis on a Primed M13 ssDNA. The reaction mixture (25 @l)

contained 30 nmi Hepes (pH 7.8), 7 nmsMgCI2,0.5 mr@iD'VF,all four dNTPs(each at 50 pM), [a-32PIdCTP (2000 cpm/pmol), 100 ng of Ml3 ssDNA(Ml3mpl8) primed with a 3-fold molar excess of a unique 17-base primer(primer 1211; New England BioLabs, Beverly, MA), and 125 @gof S-lOOextract. The reaction was incubated at 37°Cfor 1 h, and 32Pincorporation intoDNA was determined by liquid scintillation counting as described above. Toanalyze replication products, DNA was extracted with phenol:chloroform

(1:1), precipitated with ethanol, and dissolved in 20 @lof 10 mMTris-l mMEDTA (TB). The sample was subjected to electrophoresis in an alkalineagarose gel (1%) in 30 mM NaOH-l mt@iEDTA at 1.5 V/cm for 12 h. Beforebeing dried for autoradiography, the gel was fixed in 10% methanol-lO%

acetic acid. Other details were as described previously (22).

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Western Blotting. Cells were lysed in SDS-PAGE loading buffer andextracts were prepared as described above. Proteins were separated by SDSPAGEon a 12%gel andtransferredto a polyvinylidenedifluoridemembrane.Western blot analysis was performed using enhanced chemiluminescenceaccording to the manufacturer (Amersham Corp.). Anti-RP-A antibody (p34-20; generously provided as ascites fluid by Dr. Bruce Stillman, Cold SpringHarbor Laboratory, NY) was diluted 1:4000.Anti-Topo I antibody (generouslyprovided as cell supernatant by Dr. Y. C. Cheng, Yale University, New Haven,CT) was diluted 1:200.

RP-A Purification. The methodology used was essentially as described(23). Briefly, bacteria [strain BL21 (DE3)] were transformed with the expression vector plld-hRP-A containing the cDNA sequences for 70,000, 32,000,and 40,000 ofhuman RP-A (Ref. 20); a gift from Dr. Wold, University of IowaCollege of Medicine, Iowa City, IA), and a 1-literculture was grown to an A@mu of 0.6. At this point, the culture was induced with 0.2 mM isopropyl

thiogalactoside and allowed to grow for 2 h at 37°C.Cells were collected bycentrifugation and lysed by sonication in 5 ml of HI buffer [30 mM Hepes, pH7.8,1 msiDli', 0.25mMEDTA,0.25%(wlv)inositol;and0.01% (v/v)Nonidet P-40] supplemented with I mM phenylmethylsulfonyl fluoride. Thelysate was centrifuged, and the sample was loaded on an Affi-Gel blue columnequilibrated with HI buffer containing 50 mr@iKC1. The column was washedwith 50 ml each of 50 mM KC1,0.8 MKCI, and 0.5 MNaSCN. RP-A elutedwith I .5 MNaSCN. Peak fractions were loaded on a hydroxylapatite columnequilibrated with HI buffer. Protein was eluted with four column volumes ofHI buffercontaining80 mMK2PO4.The resultingpeakfractionswere appliedto a Mono-Q column equilibrated with HI buffer containing 100 mMKC1.Thecolumn was developed with a 10-mi linear KC1gradient (100—400ms@iKCI).RP-A elutes with 300 mMKC1and is homogeneous by SDS-PAGE followedby silver staining.

Topo I Purification and Activity Measurements. Topo I was purifiedfrom HeLa cell nuclei according to the method of Liu and Miller (24). Briefly,HeLa cell nuclei were lysed, and DNA was precipitated with polyethyleneglycol. The resulting supernatant was loaded on a hydroxylapatite columnequilibrated with 1 M NaCI, 50 mi@iTris (pH 7.5), 6% (w/v) polyethyleneglycol, 10 mMmercaptoethanol, and 1 mMphenylmethylsulfonyl fluoride. Thecolumn was developed with a 0.2—0.7Mpotassium phosphate gradient, and the

fractions containing Topo I activity were pooled and loaded on a phosphocellulose (P-ll) column. Topo I was eluted with 0.2—0.7Mpotassium phosphatelinear gradient, and fractions containing activity were pooled and dialyzed.Finally, active fractions were loaded on ssDNA cellulose. Topo I activityeluted in the 0.4 MKCI wash and was homogeneous by SDS-PAGE and silverstaining.

Topo I activity during purification and for the experiments described herewas measured by the relaxation of superhelical plasmid DNA (pUCl8, orpSV0ls@.EP Ref. 24). The reaction mixture contained 50 mM Tris-HC1 (pH7.5), 120 mM KC1, 10 mM MgCl2, 0.5 m@iD1T, 0.5 m@iEDTA, 30 @g/mlBSA, 20 @.&g/m1plasmidDNA, andvariousamountsof enzyme. The reactionswere incubated at 37°Cfor 30 mm and were stopped by the addition of 5 pAof5% SDS, 25% Ficoll-400, containing 0.25 mg/ml bromphenol blue. Sampleswere electrophoresed for 2 h in 1% agarose with 0.5X ThE (45 mi@iTris pH8.2,45mMboricacid,and1mMEDTA)at2V/cmandvisualizedbystainingwith ethidium bromide. One unit is defined as the amount of enzyme requiredto relax 0.5 i@gof plasmid in 30 mm at 37°C.

Measurement of DNA Replication in Intact Cells. Aliquots of cells (3.0ml) from the same spinner cultures used to prepare S-lOO cytoplasmic extractswere supplemented with 3 pCi of [3H]thymidine. Samples were incubated at37°Cfor 15 rain in an atmosphere of 5% CO2 and 95% air. Cells were thencollected and loaded on GF/A glass fiber filters, washed with 10% trichloroacetic acid and then washed with deionized water. 3H radioactivity wasmeasured using liquid scintillation counting.

PFGE. Asymmetricfield inversiongel electrophoresis,a PFGE method,was carried out in 0.5% seakem agarose (FMC Bioproducts) in 0.5X ThE at10°Cfor 40 h, by applyingcycles of 1.25 V/cm for 900 s in the directionofDNA migrationand 5.0 V/cm for 75 s in the reverse direction.Cells weretreated with CVF in dishes and embedded, after trypsinization, in agarose,where they were lysed before electrophoresis. Quantification and analysis forDNA dsbs present were carried out in a Phosphorlmager (Molecular Dynamics), or by scintillation counting. dabs were quantified by calculating the

fraction of activity released from the well into the lane, which equals the ratio

of the radioactivity signal in the lane versus that of the entire sample (well plus

lane) in CPT-treated and nontreated samples. Details of the assay have beenpublished (25).

Results

Reduced DNA Replication Activity in Extracts of CPT-treatedCells. Treatment for 3 h with 0.5 p.M CPT reduces incorporation of[3H]thymidine into DNA of HeLa cells to 18% of untreated controls,indicating a strong inhibition of DNA replication (Fig. 1A, firstcolumn). This inhibition may be due to damage that alters the substrate properties of the DNA during replication. It is known that a

collision of the advancing replication fork with the cleavable complexwill inhibit DNA replication and will lead to the formation of DNAdsbs (see “Introduction―).Indeed, exposure for 3 h of HeLa cells toCPT leads to a dose-dependent formation of dsbs that can be assayedimmediately thereafter by PFGE (Fig. 2). A comparison betweenresults of cells exposed to Cli.' and results of cells exposed to X-rays(Fig. 2), suggests that cells exposed for 3 h to 4 @.LMCPT have in their

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Fig. I . A. DNA synthesis as measured by incorporation of (3Hlthymidine in exponentially growing HeLa cells (in vivo) incubated for 3 h with 0.5 p.M CPT (first column);

results are normalized to the levels of DNA synthesis observed in nontreated cells. DNAsynthesis in vitro, using the SV4O-basedin vivo replication assay and extracts (125 @sg)prepared from HeLa cells exposed to 0.5 @MCPT for 3 h (second column); results arenormalized to the levels of DNA synthesis observed with extracts of nontreated cells. Invitro DNA replication using extracts of nontreated cells ( 125 @sg)in the presence of 0.5g.LM (third column) and 300 @M(fourth column) CPT. B, Agarose gel electrophoresis and

radioautography of the products of DNA replication reactions carried out in vitro in thepresence of extracts from nontreated or CPT-treated cells as described in A. Shown arealso results of a reaction assembled by mixing 100 @.sgof extract of nontreated cells with10 @gof extract of CPT-treated cells. c, DNA chain elongation catalyzed by extracts ofnontreated or CPT-treated cells using primed M13 DNA as a substrate. Columns, meanfrom four replicate experiments: bars, SE.

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Topo I Levels and Activity Are Unchanged in Extracts ofCPT-treated Cells. The reductionin replicationactivity of extractsprepared from CPT-treated cells may be due to an inhibition/depletionof key replication factors. Because CPT is known to specifically

interact with DNA-bound Topo I to trap the cleavable complex, we

inquired whether this effect causes an inactivation or depletion of theenzyme in the extract. Western blots for Topo I in extracts of nontreated or CPT-treated cells does not show a reduction in enzymelevels (Fig. 3A). In addition, activity measurements, using as anendpoint the relaxation of supercoiled plasmid DNA, demonstrate nochange in the overall activity in extracts from CPT-treated cells (Fig.3 B and C). Shown in the figure for comparison are also resultsobtained with extract of cells exposed to 50-Gy X-rays.

As a final confirmation that inactivation of Topo I may not underliethe reduced replication activity of extracts of CPT-treated cells, weadded purified Topo I in reactions assembled with extracts of nontreated or CPT-treated cells. The results show (Fig. 3D) that addition

of purified Topo I does not affect the replication activity of extracts ofeither nontreated or CPT-treated cells.

Evidence for Factors in Extracts of CPT-treated Cells thatInhibit DNA Replication. We inquired whether extracts of CPTtreated cells contain factors that inhibit DNA replication. For thispurpose, replicate reactions were assembled with 100 @.tgof extract of

nontreated cells, and the indicated amount of extract from CPT-treated

or nontreated cells was added. Reactions were incubated at 37°Cfor1 h to measure DNA synthesis. The results obtained are shown in Fig.4A. Addition of the extract of nontreated cells increased replicationactivity. However, addition of extract from CPT-treated cells rapidly

reduced replication activity from 23 to about 6 pmol. These resultssuggest the presence of a replication inhibitor in the extract of CPTtreated cells. The inhibitor must be a high molecular weight moleculebecause it is retained during dialysis in a membrane with a cutoff ofMr 14,000.

The Reduction in DNA Replication in Extracts of CPT-treatedCells Reflects Mainly Inhibition of Replicon Initiation. In aneffortto examine further the mechanism of inhibition, we sought information regarding the step of DNA replication preferentially inhibited.For this purpose, we examined the size distribution of newly formedDNA in the in vitro reactions. Fig. lB shows an analysis of thereplication products by gel electrophoresis followed by radioautography using a Phosphorlmager. The size distribution of newly produced(radioactivity-labeled) DNAs is similar in reactions assembled withextracts of nontreated and CPT-treated cells, despite the fact that

much less DNA is synthesized in the latter reaction. Similar results

were also obtained in reactions assembled with extract of nontreatedcells in the presence of small amounts of extract from CPT-treatedcells. These observations are compatible with an inhibition of theinitiation events of DNA replication in extracts of CPT-treated cells.

Inhibition of DNA chain elongation would have produced a morediffused pattern of products and would have increased the presence of

small DNA molecules, neither of which was actually observed.We confirmed that DNA chain elongation is not affected in extracts

of CPT-treated cells by measuring DNA chain elongation usingprimed Ml3 ssDNA and extracts of nontreated and CPT-treated cells.Cell extracts (5-100) contained all factors required for elongating aprimer on a Ml3 substrate. No reduction was observed in the abilityof extracts of CPT-treated cells to support DNA chain elongation, andactually a slight increase in this ability is reproducibly found (Fig.10. Also, the size distribution of the synthesized DNA was similar inreactions assembled with extract from nontreated and CPT-treated

cells (results not shown). In a separate experiment, we examined the

effect of CPT on DNA chain elongation using the above describedassay and extracts of nontreated cells. There was no effect of CPT on

2 4 6CPT, tM I X-rnys, Gy

Fig. 2. CPT-induced DNA dsbs in cells exposed to different concentrations for 3 h, asmeasured by asymmetric field inversion gel electrophoresis. Plotted is the fraction ofDNA-associated radioactivity (I'4C)thymidine) released from the well into the lane as afunction of drug concentration, which is a measure of the number of the dabs present.- - - -, results obtained with cells exposed to the indicated doses of X-rays and assayed

immediately thereafter.

DNA a number of dsbs approximately equivalent to that produced byan acute dose of 6-Gy X-rays. Assuming a linear correlation betweenfraction of activity released and dose, we calculate that cells exposedto 0.5 @.LMCPT will carry a load of dsbs equivalent to that of 0.8-GyX-rays. It is unlikely that this load of dsbs can, by itself, cause thelevel of replication inhibition observed in Fig. lA (—80%), as lO-GyX-rays cause a reduction in DNA synthesis by only 10—15%(26, 27).

We inquired, therefore, whether effects other than damage in theDNA substrate contribute to the inhibition of DNA replication ohserved in intact cells. In particular, we were interested in examining

whether inactivation of key replication enzymes or the activation of acheckpoint in S phase contributed to the inhibition. As a first step

towards this goal, we compared the replication activity of extractsprepared from CPT-treated cells to that of untreated controls using the

SV4O-based in vitro replication assay. We reasoned that this assay, byallowing a separation of the substrate DNA from the enzymaticmachinery (it uses undamaged plasmid DNA as a substrate, ratherthan the damaged cellular DNA of in vivo experiments), offers aunique way to investigate enzyme inactivation/depletion and regula

tory factor production as contributors to the observed down-regulationof DNA replication. The results in Fig. IA (second column) indicatethat extracts of cells exposed for 3 h to 0.5 p@ CPT show a strongreduction in their ability to support DNA replication in vitro. In fact,the level of inhibition observed in cell extracts tested in vitro (80%) iscomparable to the inhibition observed in intact cells tested in vivo(82%), suggesting that effects, in addition to damaged DNA, contribute to the DNA replication arrest observed in cells exposed to CPT.There was no evidence for apoptosis in cells treated with CPT underthe conditions used to prepare the extracts.

It is not possible to attribute part of the inhibition observed in invitro replication reactions assembled with extract of CPT-treated cellsto the presence of free CI―!'because unbound drug is removed duringextract preparation in the final dialysis step. To evaluate the directeffects of CPT on DNA replication, we tested the effect ofO.5 and 300@LMCPT on DNA replication in vitro in reactions assembled with

extract from nontreated cells. The drug was added directly to thereaction mixture and DNA synthesis was measured. The results in Fig.IA (third and four columns) confirm previous observations (7) and

suggest that CPT has only a small direct effect on DNA replication,even at concentrations 200-fold higher than those administered tocells.

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Fig. 3. A, Western blot of Topo I in extracts ofnontreated and CPT-treated cells. Protein equivalent to 2 x i0@cells was loaded per well. B, TopoI activityas measuredby the relaxationaftervanous periods of incubation of supercoiled plasmid(pSV0L@EP)in extracts of nontreated and CPTtreated cells (50 ng). Shown for comparison are alsoresults obtained from extracts of cells exposed to50-GyX-rays.C, quantitationof the resultsshownin B using a Fluorolmager. Plotted is the fraction ofplasmid DNA remaining supercoiled as a functionof the incubation time. D, in vitro DNA replicationwith extracts of nontreated and CPT-treated cells inthepresenceof variousamountof purifiedTopoI.

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chain elongation when it was present in the reaction mixture either at0.5 or 300 p.M (results not shown).

RP-A as a Modulator of DNA Replication in Extracts of CPTtreated Cells. RP-A plays an essential role in the initiation of in vitroDNA replication (28—30).Furthermore, RP-A p34 phosphorylationhas been demonstrated following exposure to DNA-damaging agents,and a correlation between this phosphorylation and its ability tosupport DNA replication has been proposed (31—33).Therefore, weexamined RP-A phosphorylation and possible inactivation in CPT

treated cells. Fig. 4B shows that in cells treated for 3 h with 0.5 ,.LMCPT, approximately 30% of RP-A p34 migrates slower and is therefore likely to be phosphorylated. RP-A phosphorylation is partly lostduring extract preparation, probably as a result of phosphatase action(results not shown). Because this phosphorylation of RP-A p34 or

other undetectable posttranslational modifications of the RP-A complex may affect its DNA strand binding activity and, as a result, itsability to support DNA replication, we inquired whether addition ofrecombinant RP-A can restore replication activity in extracts of CPTtreated cells. The results (Fig. 4C) indicate that addition of increasingamounts of RP-A does not overcome the replication inhibition observed in extracts of CPT-treated cells. Similar results were obtainedwhen RP-A was added to a mixture of extracts from CPT-treated andnontreated cells. These results suggest that the inhibition observed inDNA replication is unlikely to derive directly from RP-A inactivation.

Discussion

If fork collisions with cleavable complexes, as predicted by the“forkcollision model,―were the only mechanism by which CPTinhibited DNA replication, damage/alterations in the substrate DNAwould be the main cause of the inhibition. Thus, in an experimentdesigned to separate the damaged DNA substrate from the enzymaticmachinery, as is possible using the SV4O in vitro replication assay,one would expect levels of replication of plasmid DNA comparable tothose observed with extracts of non-treated cells. However, the results

presented in Fig. lA suggest that extracts prepared from CPT-treatedcells are significantly impaired in their ability to replicate plasmid

DNA. Indeed, the level of replication inhibition observed in vivo isvery similar to that observed in vitro, suggesting that the determinantsof inhibition are the same under both conditions.

The known effects of CPT on Topo I (1—3)and the fact that thisenzyme is essential for in vitro DNA replication (34) would suggest

that the reduction observed in the in vitro replication activity ofextracts of CPT-treated cells derives from a reduction in Topo Iactivity. Indeed, exposure of cells to DNA-damaging agents has been

reported to reduce Topo I activity, probably via a posttranslationalmechanism (35). Nevertheless, our results (Fig. 3, A—C)demonstrate

that neither the levels nor the activity of Topo I was reduced inextracts of CPT-treated cells and that the addition of purified, activeTopo I did not restore replication activity.

We hypothesize, therefore, that the reduced DNA replication activity of extracts prepared from CPT-treated cells is due to the formation or activation of a regulatory molecule(s) that inhibits DNAreplication in vivo and in vitro. The activation of such a regulator of

DNA replication would reflect the activation of a surveillance mechanism in response to DNA replication inhibition or DNA damageproduction and will be equivalent to the activation of a checkpoint inS (14 —16).Several observations suggest that the effects of CPT onDNA replication and cytotoxicity may be determined by effects inaddition to those directly deriving from DNA damage. First, theamount of DNA damage (dsbs in our experiments) present in CPT

treated cells is relatively low and cannot explain the level of DNAreplication inhibition observed (Figs. IA and 2). Second, DNA replication per se is rather resistant to CPT exposure, as indicated byexperiments in which 300 @tMCPT was added to reactions measuringreplication in vitro (Ref. 7; results in Fig. IA). This would suggest thatthe inhibition observed in vivo at CPT concentrations as low as 0. 1 ,.tMmay not reflect solely a direct inhibition of the replication machinery,but rather the combination of a direct DNA damage-related inhibition

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Fig. 4. A, DNA replication activity in vitro inreactions assembled with 100 @igof extract of nontreated cells and the indicated amounts of extractprotein from either nontreated or CPT-treated cells.B, Western blot for RP-A p34 from nontreated andCPT-treated cells. C, DNA replication activity invitro in reactions assembled with 100 @gof extractof nontreated or CPT-treated cells and the indicatedamounts of recombinant RP-A. Shown are also theresults of reactions assembled with 100 .sgof cxtract of nontreated cells and 10 @gof extract ofCPT-treated cells.

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limited to chain elongation (7). It is particularly interesting in thisregard that chain elongation activity is not inhibited in extracts ofCPT-treated cells (Fig. 1C). The nature of the DNA replication

inhibitor is presently unknown but it seems unlikely to be RP-Abecause inactivation of this replication factor would not be expectedto be dominant in mixing experiments. More importantly, recombinant RP-A fails to restore DNA replication activity (Fig. 4C). Weobserved phosphorylation of RP-A p34 in response to CPT treatment(Fig. 4B), suggesting the operation of regulatory processes on theprotein. It has been reported that throughout the cell cycle, p34cdc2kinase, in association with cyclin A or B, phosphorylates RP-A p34 atSer-23 and Ser-29 (37). Other reports indicate that RP-A p34 isphosphorylated in two steps, first by cdk-cyclin A and then byDNA-PK, and that@ prevents this phosphorylation incyclin A-activated extracts of G1 cells (38). DNA-PK has been purifled as the kinase responsible for RP-A p34 phosphorylation during invitro replication (39). However, no direct association between RP-Ap34 phosphorylation and DNA replication activity could be established (38—41). Ionizing radiation-induced phosphorylation of RP-Awas found to be lacking in scid cells known to be deficient inDNA-PK (42), but these results are not confirmed by recent reports(43). Thus, the RP-A kinase responding to DNA damage remains tobe identified. The role of RP-A p34 phosphorylation in RP-A activity

and an indirect inhibition induced by the activation of a checkpoint.The small direct effect of CPT on DNA replication in vitro and thestrong inhibition observed in vivo and in extracts prepared from

CPT-treated cells would actually suggest that the indirect mechanism

dominates the response. Third, inhibition of DNA replication inCPT-treated cells usually persists for a long time after drug removal

and does not correlate with the presence of DNA damage or thetrapping of Topo I-DNA cleavable complexes (13), which againsuggests the activation of checkpoints.

Several lines of investigation suggest the activation of checkpointsin cells exposed to CPT. Cells treated with CPT are blocked in G2, anindication of the activation of a checkpoint in this phase of the cellcycle (1—3,12). Recently, evidence has been presented that CPTcytotoxicity may be affected by the ability of cells to activate check

points in S and G2 (13). Given the specificity of CPT action onS-phase cells, it seems reasonable that these cells will mount an activeresponse after such treatment to minimize its adverse effects. Such aresponse may be initiated by the DNA damage produced and mayhave important consequences in overall cytotoxicity (36).

Our results suggest that DNA replication is regulated in CPTtreated cells by a dominant factor that specifically inhibits the initiation steps of DNA replication. This mode of inhibition is differentfrom that associated with the direct effect of CPT that is thought to be

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remains uncertain, and our results suggest that RP-A may not be

contributing to the replication inhibition observed in extracts of CPTtreated cells. However, indirect effects of RP-A on the regulation ofDNA replication cannot be excluded.

In summary, we have presented evidence for factors inhibitingDNA replication in extracts of CPT-treated cells. We showed thatthese factors act in a dominant fashion and that they specificallyinhibit replicon initiation; their effects are distinct from the directeffect of CPT on chain elongation. We propose that these factors areinduced or activated in cells in response to CPT-induced DNA damage and that they act in a similar fashion in the intact cell in vivo.Inhibition of replicon initiation in CPT-treated cells will reduce theDNA relaxation requirements and thus the interactions between TopoI and DNA, on the one hand, and the interaction between trappedcleavable complexes and replication forks, on the other hand. Bothphenomena have the potential to reduce the toxic consequences ofCPT treatment. We propose that these factors are the effectors of anS-phase checkpoint and that they are important determinants of drugtoxicity. The focus of future research will be the identification andcharacterization of these factors.

Acknowledgments

The authors are indebted to Drs. B. Stillman, R. E. Lanford, J. Hurwitz,Y. C. Cheng, andM. S. Wold for reagentsandto N. Mottfor secretarialhelpand for editing the manuscript.

References

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