Protein-Linked DNA Strand Breaks Induced in Mammalian Cells by Camptothecin, an Inhibitor of Topoisomerase I Joseph M

(CANCER RESEARCH 49, 5016-5022. September 15. 1989] Protein-linked DNA Strand Breaks Induced in Mammalian Cells by Camptothecin, an Inhibitor of Topoisomerase I Joseph M. Covey,1 Christine Jaxel, Kurt W. Kohn, and Yves Pommier

Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20S92

ABSTRACT tion of SDS, Camptothecin has been used to localize topoisom erase I binding sites in mammalian genes (10-13). However, it Camptothecin was recently identified as an inhibitor of mammalian has also been reported that a significant fraction of the breaks topoisomerase I. Similar to inhibitors of topoisomerase II, Camptothecin produces DNA single-strand breaks (SSB) and DNA-protein cross-links produced by Camptothecin in mammalian cells are not tightly (DPC) in mammalian cells. However, their one-to-one association, ex linked to protein (14) and that Camptothecin can produce DNA pected for trapped topoisomerase complexes, has not previously been cleavage without topoisomerase I in the presence of UV light demonstrated. We have studied camptothecin-induced SSB and DPC in and copper (15). These reports raised some questions as to Chinese hamster DC3F cells and their isolated nuclei, using the DNA whether camptothecin-induced DNA breaks in cells result alkaline elution technique. It was found that the SSB and DPC frequen solely and specifically from inhibition of topoisomerase I. cies detected following Camptothecin treatment depend upon the condi The present study was undertaken in order to reassess the tions used for lysis. When lysis was with sodium dodecyl sulfate, the question of protein linkage of DNA breaks produced by camp- observed frequencies of SSB and DPC were 2- to 3-fold greater than tothecin in mammalian cells and to determine the frequency of when sodium dodecyl sarkosinate (Sarkosyl) was used. In either case, the DNA lesions (SSB, DSB, and DPC) induced by camptothe- the SSIf :!)!â€¢(ratiowas close to 1. All of the camptothecin-induced SSB cin under various conditions. The DNA filter elution method were protein linked, as indicated by the absence of DNA elution under ology, which originally detected the production of protein- nondeproteinmng conditions. DNA cleavage assays with purified topoi somerase I also indicated that the weaker Sarkosyl detergent fails to trap linked strand breaks in response to inhibitors of topoisomerase all of the enzyme-DNA complexes. In contrast, lysis conditions had little II (16, 17), is the means by which these lesions can best be effect on levels of SSB or DPC produced by 4'-(9-acridinylamino)- detected and quantified in mammalian cells (18). To define the methanesulfon-/n-anisidide, suggesting that trapping of topoisomerase II protein linkage of DNA breaks expected in the case of topoi complexes occurs equally well with either detergent. In experiments using somerase action, one must (a) demonstrate the production of isolated nuclei, it was found that the camptothecin-induced SSB, in SSB, (b) demonstrate the production of DPC, (c) show that contrast to trapped topoisomerase II complexes, can form and reverse within minutes at 4Â°C.The activity of Camptothecin at low temperature SSB and DPC are produced in approximately equal numbers, and (d) show that the induced SSB and DPC are localized with was also seen with purified topoisomerase I. These results support the respect to each other (19). hypothesis that the SSB and DPC induced by Camptothecin in mamma The results of the present study, contrary to previously pub lian cells are due to an action on topoisomerase I. lished reports, show that essentially all of the DNA strand breaks produced by Camptothecin in a Chinese hamster cell line INTRODUCTION (DC3F) are protein linked and that SSB and DPC are within experimental error of a 1:1 ratio. This is consistent with the Camptothecin (Fig. 1) is an alkaloid that is isolated from hypothesis that camptothecin-induced SSB and DPC represent Camptotheca acuminata, a tree indigenous to mainland China drug-stabilized topoisomerase I complexes. (1). Camptothecin is an active antitumor agent in a number of In the course of these studies, it was found that camptothecin- murine systems (2) and current drug development efforts are induced DNA breaks had two unusual characteristics: (a) their focused on the synthesis of Camptothecin analogues that could detection was dependent upon the detergent used for cell lysis be used in cancer chemotherapy (3). and (b) they could form and reverse at 0Â°C.Similar properties Earlier observations showed that treatment of mammalian were also found with purified mouse leukemia L1210 topoisom cells with Camptothecin results in a reversible fragmentation of erase I. These results provide evidence for the involvement of DNA (4, 5), a potent inhibition of DNA and RNA synthesis topoisomerase I inhibition as the sole mechanism for the in (6, 7), and a subsequent impairment of cell division (4). It has duction of DNA breaks by Camptothecin in mammalian cells. recently been demonstrated that Camptothecin is a specific inhibitor of purified mammalian topoisomerase I and that this inhibition results from the stabilization of topoisomerase I- MATERIALS AND METHODS DNA complexes in which one enzyme molecule is covalently bound to the 3'-terminus of a SSB- (8, 9). Drugs and Chemicals. Camptothecin (NSC 94600) was a gift from Because Camptothecin is currently the only known topoisom Dr. Monroe Wall, Research Triangle Institute (Research Triangle Park, erase I inhibitor that traps topoisomerase I-DNA covalent NC). m-AMSA (NSC 249992) was obtained from the Pharmaceutical complexes and because these complexes can be converted to Resources Branch, Division of Cancer Treatment, National Cancer DNA breaks in purified enzyme-DNA systems upon the addi- Institute (Bethesda, MD). Stock solutions of both drugs were prepared in dimethyl sulfoxide at 10 HIM.[fntf/i>'/-3H]Thyrnidine, [2-14C]thymi- Received 2/9/89; accepted 6/15/89. dine (specific activity, 20 and 0.05 Ci/mmol, respectively), and [a-"P]- The costs of publication of this article were defrayed in part by the payment dGTP were purchased from NEN Research Products (Boston, MA). of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. SV40 DNA and Banl and ///Â»allrestriction endonucleases were pur ' To whom requests for reprints should be addressed, at Pharmacology Branch. chased from BRL (Gaithersburg. MD). Developmental Therapeutics Program, Division of Cancer Treatment, National Cell Culture, Radiolabeling, and Drug Treatments. DC3F Chinese Cancer Institute. Executive Plaza North. Room 841. Bethesda, MD 20892. hamster lung fibroblast cells (20) were grown in monolayer cultures in 'The abbreviations used are: SSB, DNA single-strand break; DPC. DNA- Eagle's modified minimal essential medium that was supplemented protein cross-link; DSB, DNA double-strand break; LDS, lithium dodecyl sulfate: m-AMSA, 4'-(9-acridinylamino)-methanesulfon-m-anisidide; SDS. sodium do with 10% heat inactivated fetal calf serum plus 2 IMMglutamine, 1 HIM decyl sulfate. sodium pyruvate, 0.1 m\r nonessential amino acids, penicillin-strepto- 5016

(no SDS) at a flow rate of 0.02-0.03 ml/min. Fractions were collected at 3-h intervals for 15 h. DPC frequencies were calculated according to the bound-to-one terminus model (16): />, = [(1 - r)-1 - (1 - r0)-<]PB

where r0 and r are the retentions on the filter of DNA from [MC]- thymidine-labeled control and treated DC3F cells, respectively, and Pa is the radiation dose administered (3000 rad). The degree to which r exceeds r0 is a measure of DPC frequency, and the results (/â€¢â€ž)are expressed in rad-equivalents. Demonstration of the Protein Linkage of DNA Breaks. Breaks not tightly bound to protein (frank breaks) were detected by alkaline elution that was carried out under DNA-denaturing, nondeproteinizing condi

Fig. 1. Structure of 20-(5>camptothecin. Et, ethyl. tions as described for the DPC assay, except that treated cells received no irradiation prior to elution. Elution rates were compared with those mycin (50 units/ml and 50 Mg/ml, respectively), and 20 mM N-(2- from 7-irradiated control cells. hydroxyethyl)piperazine-Ar'-(2-ethanesulfonic acid) (all from Advanced Determination of DNA Double-Strand Break Frequency. DSB were Biotechnologies, Inc., Columbia, MD). Suspension cultures of L1210 analyzed using DNA nondenaturing alkaline elution (pH 9.6) that was cells were maintained as previously described (21). For alkaline elution carried out under deproteinizing conditions (22, 24). No more than 2 experiments, DC3F cells were seeded at 1 x IO5cells/25-cm2 flask in x IO5 DC3F cells and no internal standard cells were layered onto medium containing 0.02 ^Ci/ml [14C]thymidine and were grown for 24 polycarbonate filters. The outlets of the funnels were connected to a h. Cells were then washed with unlabeled medium and incubated for an peristaltic pump prior to cell lysis with 2 ml of SDS lysis solution that additional 16-24 h prior to drug treatment. 1.1210 cells, which were contained 0.5 mg/ml proteinase K. DNA was eluted with tetrapro used for internal standards in elution experiments, were labeled for 16- pylammonium hydroxide-EDTA, 0.1% SDS, pH 9.6, and collected in 24 h with 0.2 i<(Ã¯/nil[â€¢'!Ijthymidincand chased in unlabeled medium 3-h fractions for 15 h. DSB were quantitated by comparing the elution for at least 2 h prior to use. Camptothecin or m-AMSA solutions were rates of DNA from treated cells with those from non-drug-treated cells added to DC3F cultures or suspensions of isolated nuclei such that the which had been irradiated with various doses (2,000-10,000 rad) of y- concentration of dimethyl sulfoxide never exceeded 2%. Unless other radiation. wise indicated, drug exposures were for 30 min at 37Â°C,and drug Kinetics of Camptothecin-induced SSB Formation and Reversal at treatments were terminated by rinsing cells twice with cold Hanks' 4Â°C.Isolated nuclei were carefully deposited onto polycarbonate filters balanced salt solution and then scraping them into the same buffer. in smokestack-type elution funnels, which were maintained at 4Â°C Preparation of DC3F Nuclei. The procedure for isolation of DC3F throughout the experiment. Drug treatments were initiated by allowing nuclei has been described previously (22). Briefly, DC3F cells were rinsed, scraped into 4"C nucleus buffer [150 mM NaCl, 1 mM KH2PO4, 3 ml of cold nucleus buffer that contained the desired camptothecin concentration to drip through the filter. The time course of SSB 5 mM MgCl2, 0.1 mM dithiothreitol, and 1 mM ethylene glycol bis(/3- aminoethyl ether)-yV^V^V'^V'-tetraacetic acid], and centrifuged [460 x formation was followed by lysing nuclei at various times with either SDS lysis solution plus proteinase K or a comparable solution (LDS g, 6 min). Cells were resuspended in 0.1 volume of cold nucleus buffer; lysis) prepared with 1% LDS instead of 2% SDS. LDS is more soluble 0.9 volume of cold nucleus buffer that contained 0.3% Triton X-100 was then added and the mixture was gently rotated for 8 min at 4Â°Cin than SDS at reduced temperatures. The rate of SSB reversal was determined by lysing the nuclei at various times after they had been the dark. The nuclei were pelleted by centrifugation (460 x g, 10 min) treated for 30 min and then washed with fresh nucleus buffer. Alkaline and resuspended in nucleus buffer at the indicated temperature. elution was performed as described above. Determination of DNA Single-Strand Break Frequency. SSB were Topoisomerase I-mediated Cleavage of Supercoiled DNA. SV40 DNA analyzed using DNA-denaturing (pH 12.1) alkaline elution that was carried out under deproteinizing conditions (17, 23). Briefly, [I4C]- (0.1 Â¿/g)waspreincubated for 15 min in 20 ^1 of 0.01 M Tris-HCl-0.05 M KC1-5 mM MgCb-O.l mivi EDTA-15 jig/ml bovine serum albumin, thymidine-labeled DC3F cells or isolated nuclei were layered onto pH 7.5, in the presence or absence of 10 JIM camptothecin. Purified polycarbonate filters (2.0 Â¿im;NucleoporeCorp., Pleasanton, CA). Cells LI 210 topoisomerase I (25) (45 units) was then added. Reaction or nuclei were lysed either with a solution containing 2% SDS, 0.1 M mixtures were incubated for 10 min at 37"C and were stopped as glycine, 0.025 M disodium EDTA, and 0.5 mg/ml proteinase K, pH 10 indicated in the legend to Fig. 3, followed by an additional 60 min (SDS lysis) or, in a few experiments, with LS-10 (2 M NaCl, 0.2% incubation at 37"C. Reaction mixtures were loaded onto a 1% agarose Sarkosyl, and 0.04 M disodium EDTA, pH 10) that contained 0.5 mg/ gel that contained 2 Mg/ml chloroquine and were run at 1.4 V/cm for ml proteinase K (see below). The lysis solution was washed from the 15 h. The gel was stained with ethidium bromide (2 //\i). destained filters with 5 ml of 0.02 M EDTA, pH 10, and the DNA was eluted with tetrapropylammonium hydroxide-EDTA, 0.1% SDS, pH 12.1, at with 1 mM MgSO4 and photographed under UV light. The photo a flow rate of 0.08-0.12 ml/min. Fractions were collected at 5-min graphic negatives were scanned with a Beckman DU-8B spectropho- intervals for 30 min. In some experiments, the more sensitive overnight tometer that was connected to a computer, in order to determine the elution method, in which the flow rate is 0.02-0.03 ml/min and 3-h area under the absorbance curves and to quantify DNA nicking (26). Topoisomerase I-mediated Cleavage of "P-end Labeled SV40 DNA. fractions are collected, was used. Fractions and filters were processed 32P-3'-end labeled SV40 DNA (0.04 Mg)(Banl-Hpall fragment) (26) and radioactivity was determined as previously described (23). Drug- induced SSB frequencies were expressed as the ->-radialion dose which was incubated in 30 ii\ of reaction buffer with 220 units of LI210 would produce the same elution rate (rad-equivalents). topoisomerase I for 30 min at the indicated temperatures, in the Determination of DNA-Protein Cross-Link Frequency. Nondeprotein- presence of absence of camptothecin. Reactions were stopped by adding izing, DNA-denaturing, alkaline elution in "smokestack-type" elution 1% SDS, 20 mM EDTA, and 0.5 mg/ml proteinase K, with an addi tional incubation for 60 min at 37Â°C.After extraction with phenol- funnels (23) was used to determine DPC frequency (16). Aliquots containing approximately 5 x 10s [14C]thymidine-labeIed drug-treated chloroform and precipitation with ethanol, the DNA was resuspended or control cells were irradiated on ice with 3000 rad prior to elution. in 2.5 Â¿ilMaxam and Gilbert loading buffer (80% formamide, 10 mM Cells were gently layered onto polyvinylchloride-acrylic copolymer NaOH, 1 mM EDTA, 0.1% xylene cyanol, and 0.1% bromophenol filters (Metricel DM-800; Gellman Sciences, Ann Arbor, MI) and lysed blue). After heating at 80-90"C for 2 min, equal counts were loaded with either LS-10 or SDS lysis solution. The lysis solution was rinsed onto a 6% Maxam-Gilbert sequencing gel, which was run at 2900 V away with 5 ml of 0.02 M EDTA (pH 10), and the DNA was eluted (80 W) for 75 min. The gel was transferred to a No. 3MM paper sheet, from the filter with tetrapropylammonium hydroxide-EDTA, pH 12.1 dried, and autoradiographed with Kodak XAR-5 film. 5017

RESULTS

Effect of Lysis Conditions on Camptothecin-induced DNA Breaks. Representative alkaline elution curves from SSB exper 1 23456 789 10 11 iments using several types of lysis solutions are shown in Fig. 2. /H-AMSA was used as a control because its cellular effects have been extensively characterized by alkaline elution. The slopes of the elution curves of DNA from /w-AMSA-treated Rei cells were approximately the same following lysis with SDS lysis solution or LS-10 over a range of NaCl concentrations. Se However, after LS-10 lysis the w-AMSA elution curves dem Topo I - + + ++ + + + + + + onstrated a significant nonlinearity. This may result from a CPT (10 (Â¿M) - - + - + - + - + - + pool of more slowly eluting DNA as a consequence of incom plete deproteinization by LS-10/proteinase K lysis. In contrast, Stop Condition A B C D E the elution rate of DNA from camptothecin-treated cells was markedly less following LS-10 than after SDS lysis. These results suggest that camptothecin-induced SSB are better de B tected using SDS lysis than LS-10. The differential effects of the two types of lysis solutions upon camptothecin-induced topoisomerase I-mediated DNA lesions was further investigated in a purified system (Fig. 3). Cleavage of supercoiled SV40 DNA was measured following incubation with camptothecin and purified LI210 topoisomer ase I under different detergent stop conditions. In agreement 11 with what was seen in cells, markedly less DNA cleavage was O 0.2 0.4 0.6 0.8 1 detected after stopping the reaction with Sarkosyl than with Nicked DNA (relative area) SDS. This difference was independent of the NaCl concentra Fig. 3. Effects of reaction stop conditions on camptothecin-induced topoisom tion in the Sarkosyl solution, an effect also observed in intact erase I-mediated cleavage of SV40 DNA. DNA was incubated with topoisomerase I (Topo I) in the presence or absence of camptothecin (CPT), as described in cells (Fig. 2). "Materials and Methods." Reactions were stopped with 20 mM EDTA. 0.5 mg/ Protein Linkage of Camptothecin-induced DNA Breaks in ml proteinase K, and either 1% SDS (Condition A). 2% SDS (Condition B), 0.2% DC3F Cells. Previous investigations (8) have demonstrated that Sarkosyl + 1 M NaCl (Condition C). 0.2% Sarkosyl + 0.5 M NaCl (Condition D), or 0.2% Sarkosyl with no NaCl (Condition E). The mixtures were run in a SSB produced by camptothecin in the presence of purified 1% agarose-chloroquine gel (A). The gel was photographed in UV light and the topoisomerase I are protein linked. To support the hypothesis negative was scanned spectrophotometrically to determine the areas under the that the DNA breaks produced by camptothecin in mammalian absorbance curve for each lane. The amount of nicked DNA (N) was expressed as a fraction of the value obtained with \% SDS (B). Sc, supercoiled DNA; Rei, cells are mediated by topoisomerase I, it was therefore impor relaxed topoisomcrs. tant to demonstrate their protein linkage. First, SSB and DPC frequencies were quantified after camp tothecin treatments. As shown in Fig. 4, when both assays were Type of Elution performed with SDS lysis, SSB and DPC frequencies were SSB (SDS Lysis) â€¢¿DPC(SDS Lysis) nearly equal over a camptothecin concentration range from 0.1 â€¢¿FrankBreaks SSB (LS10 Lysis) DPC (LS10 Lysis)

D Camptothecin (,,M) O Fig. 4. Dose response for camptothecin-induced SSB and DPC in DC3F cells. Cells were treated with the indicated concentrations of camptothecin for 30 min and then the drug was removed by rinsing with cold Hanks' balanced salt solution. SSB were determined by deproteinizing alkaline elution using SDS or LS-10 lysis o and DPC were determined by nondeproteinizing alkaline elution, again with S either SDS or LS-10 lysis, as indicated. Frank breaks (i.e., breaks revealed under nondeproteinizing conditions) were determined under the conditions of the DPC assay, using LS-10 lysis but with no irradiation of drug-treated cells. Results with SDS are mean of 5-8 experiments, and LS-10 results are mean of 3-5 experi ments. Frank break data are from 2 experiments.

to 50 UM, with a plateau near 700 rad-equivalents. If DPC Fraction of [3H]DNA on Filter frequency using SDS lysis is plotted against the corresponding Fig. 2. Comparison of SDS and LS-10 lysis on the alkaline elution detection of SSB induced by camptothecin or m-AMSA. DC3F cells were treated with 2 value for SSB with this detergent, all the points fall near the fiMcamptothecin (Jilted symbols) or 2 /IM m-AMSA (open symbols) for 30 min, theoretical 1:1 equivalence line (Fig. 5). When LS-10 was used rinsed with cold drug-free Hanks' balanced salt solution, and prepared for alkaline as the lysis solution, SSB and DPC frequencies again were elution. Representative elution curves are shown for experiments performed with SDS lysis solution (Â«.O)or with LS-10 containing no NaCl (T.V), 0.5 M NaCl nearly equal, but their magnitude was approximately 2-fold (A,A), or 2 M NaCl (â€¢ I. lower compared with SDS lysis (Fig. 4). The dependence of 5018

1000 with ice-cold nucleus buffer prior to alkaline elution, they displayed evidence of additional DNA breakage, which was 800 1:1 proportional to the Camptothecin concentration. DPC forma tion could also be demonstrated in internal standard cells which had been added to cold-diluted, drug-treated nuclei (data not 600 shown). As a result, internal standards could not be used for SSB and DPC calculations in DC3F nuclei. However, the 400 interesting possibility emerged that the SSB and DPC observed in the internal standard cells were due to their treatment by 200 Camptothecin and, therefore, that Camptothecin was active at ice temperature. Formation and Reversal of Camptothecin-induced Topoisom- 200 400 600 800 erase I-mediated DNA Breaks in Isolated IK VU Nuclei at 4"( '. SSB (rad-equlvalents) To test the hypothesis that camptothecin-induced DNA lesions Fig. 5. Relationship between SSB and DPC frequencies in camptothecin- treated DC3F cells with drug washout after 30 min. SSB and DPC results from can form and reverse at ice temperature, the kinetics of camp alkaline elution experiments using SDS lysis (Fig. 4) were replotted to demon tothecin-induced SSB formation and reversal were investigated strate the correlation between lesion frequencies under these conditions. in isolated nuclei at 4Â°C(Fig. 7). Upon Camptothecin addition, SSB formed rapidly at 4Â°Cand reached a dose-dependent 2000 plateau level within 2-5 min. Upon Camptothecin removal, the SSB reversed within 20 min at 4Â°C(Fig. 7). This rapid revers ibility in the cold probably explains why lower SSB frequencies were observed in nuclei which had been treated with camptothecin at 37Â°Cand then diluted in cold nucleus buffer immedi ately prior to elution (Fig. 6). The temperature dependence of topoisomerase I-mediated cleavage of isolated SV40 DNA produced by Camptothecin was then studied, in order to correlate the effects seen in isolated nuclei with those induced by purified topoisomerase I. Fig. 8 10 100 shows the intensity of the major Camptothecin cleavage site in Camptothecin (p M) SV40 as a function of the time and temperature of the reaction. Fig. 6. Dose response for camptothecin-induced SSB in isolated DC3F nuclei. DNA cleavage was greater at 0Â°Cand 23Â°Cthan at 37Â°C.The Nuclei, prepared as described in "Materials and Methods" were treated with the lower intensity of cleavage at 37Â°Cwas confirmed in a time- indicated concentrations of Camptothecin for 30 min. Aliquots were then diluted 1:10 into cold nucleus buffer and SSB frequences were determined by alkaline course experiment, in which maximal cleavage occurred at 10 elution. min and subsequently declined (data not shown). This may be explained by degradation of the enzyme and/or Camptothecin at 37Â°C. DNA lesion frequency measurement on the detergent used is thus similar in cells and in a purified enzyme system. Camptothecin-induced DNA Breaks in DC3F Cells without Clearly, it is essential to use the same detergent lysis solution Drug Washout. The reversibility of camptothecin-induced SSB for both the SSB and DPC assays, since the efficiency of DNA in the cold suggested that the experimental design used in lesion detection is greater using SDS lysis than with LS-10. earlier experiments with DC3F cells (i.e., terminating drug Therefore, the SDS lysis solution (or LDS lysis) was used for treatments by rinsing with cold buffer) underestimated the DNA both SSB and DPC assays in all subsequent experiments. lesion frequencies. We therefore reexamined the SSB dose A second test of the protein linkage of camptothecin-induced response with cells that were exposed to constant Camptothecin DNA breaks is the absence of significant DNA elution when concentrations until the moment of lysis (Fig. 9). The results assayed in the absence of proteinase K. Camptothecin did not were qualitatively similar to those seen with washed cells (Fig. produce any DNA frank breaks (strand breaks not linked to protein), as shown by the lack of DNA elution under nondepro- 1600 teinizing conditions (Fig. 4). Camptothecin-induced DNA Breaks in Isolated 1X31 Nuclei. The activity of Camptothecin in isolated DC3F cell nuclei was first investigated by incubating suspensions of DC3F nuclei with the drug at 37Â°Cfor 30 min. As in the case of topoisomerase II inhibitors (22, 27), the reactions were stopped by 1:10 dilution in cold nucleus buffer, followed immediately by alkaline elution. In this system, camptothecin-induced SSB rose to approximately 1800 rad-equivalents at high drug concentra tions (Fig. 6), in contrast to the dose-response curve in cells (Fig. 4), which reached a plateau near 700 rad-equivalents. It Time (min) should be noted, however, that at 0.1 fiM there were significant Fig. 7. Camptothecin-induced SSB formation and reversal in isolated DC3F levels of SSB detected in cells but not in nuclei. nuclei at 4*C. Nuclei were layered onto elution filters and treated with campto- In the course of the experiments with isolated nuclei, an thecin at the indicated concentrations in situ, and then SDS (or LDS) lysis buffer unusual behavior of the internal standard cells was observed. was added at various times to terminate drug exposure. Additional nuclei were treated with Camptothecin for 30 min, rinsed with cold nuclei buffer, and lysed at When irradiated internal standard L1210 cells were added to the indicated times after drug removal. SSB frequencies were determined by camptothecin-treated DC3F nuclei which had been diluted 1:10 alkaline elution. Data shown are from two independent experiments. 5019

1.0

0.8

^ 0.6

0.4

< a Temperature (Â°C) -37- B 0 23 37 c Topo I o I Â°-2 CRT (10 M.M)

Fig. 8. Temperature dependence of camptothecin-induced, topoisomerase I- 12 18 medialed breaks in SV40 DNA. End-labeled SV40 DNA was incubated with Hours of Elution purified L12IO topoisomerase I (Topo I) in the presence or absence of 10 ^M Fig. 10. Alkaline elution curves from DSB assays of camptothecin-treated camptothecin (CPT) for 30 min at the indicated temperatures. Reactions were stopped, and the DNA was extracted and prepared for sequencing gel electropho- cells. DC3F cells were treated with camptothecin [1 JJM(â€¢),5/IM (A), or 10 >

2000 DNA in the lysis fraction, secondary to the presence of drug- induced DSB. This phenomenon has been previously observed with epipodophyllotoxin inhibitors of topoisomerase II (28, 29). In the present studies, DPC frequency was equal within a factor of 2 to the SSB values shown in Fig. 9, for camptothecin > 1000 concentrations up to 0.2 MM.However, above 0.5 MMcampto Treatment Conditions â€¢¿37C SOS Lysis thecin, DPC frequency increased rapidly, reaching a value of 4 c LOS Lysis 8703 Â±1447 rad-equivalents at 10 MM(data not shown), while 4Â°CSOSLysis SSB frequency plateaued near 1500 rad-equivalents (Fig. 9). These observations indicate that, at camptothecin concentra tions where the SSB:DPC ratio is markedly <1, a significant 0.1 1 10 percentage of DNA appears in the lysis fraction of the SSB Camptothecin assay, and DSB becomes detectable by neutral elution at higher Fig. 9. Dose response for camplothecin-induced SSB in DC3F cells with no concentrations. Therefore, for SSB frequencies above 700-1000 drug washout. Cells were treated with various concentrations of camptothecin for rad-equivalents, SSB elution underestimates the true protein- 30 min at the indicated temperatures and were loaded onto elution filters without removal of the drug. SSB were determined by DNA-denaturing, deproteinizing, linked strand break frequency and, thus, DPC elution is a more alkaline elution. accurate method to measure camptothecin-induced topoisom erase I-DNA complexes. 4). However, the SSB plateau was observed at much lower The ratio of true DNA single-strand breaks (i.e., those SSB concentrations and the unwashed cells showed SSB frequencies which are not part of a DSB) to double-strand breaks produced more than 2-fold higher than the washed cells, indicating that has been determined previously for various topoisomerase II drug removal allowed a partial reversal of the breaks, even at inhibitors (17, 19) and was found to range from essentially 0 4"C. The data in Fig. 9 also demonstrate that camptothecin- for ellipticine and 5-iminodaunorubicin (compounds which pro induced SSB form to a similar extent in DC3F cells at 37Â°C duce almost exclusively DSB) to around 5 from m-AMSA. and 4"C and that SDS lysis and LDS lysis solutions are equally When the present data with camptothecin were analyzed in this effective in converting topoisomerase I-DNA cleavable com way (assuming DPC frequency is the best measure of total SSB plexes into alkaline elution-detectable DNA strand breaks. frequency), the ratio of true single- to double-strand breaks DNA Double-Strand Breaks and Summary of the DNA Le ranged from 44 to 83 (at 10 and 5 MMcamptothecin, respec sions Produced by Camptothecin in DC3F Cells. In the course tively). Thus, while inhibitors of topoisomerase II generally of conducting the SSB experiments described above, it was produce fewer true single-strand breaks per double-strand break observed that camptothecin treatments resulted in a dose-de than does 7-radiation [ratio <23 (Ref. 30)], the interaction of pendent increase in the amount of DNA appearing in the lysis camptothecin with topoisomerase I results in a single- to dou fraction. At 10 MMcamptothecin, approximately half of the ble-strand break ratio at least 2-fold greater than that of y- total DNA appeared in the lysis. Since this behavior is indicative radiation. These data indicate that camptothecin produces pre of the presence of DSB, pH 9.6 elutions were performed (Fig. dominantly SSB in mammalian cells, although DSB can be 10). Camptothecin at 5 and 10 ;<\i induced 1944 and 3762 rad- detected at high camptothecin concentrations. equivalents of DSB, respectively. No DSB could be detected at Using optimized experimental conditions, the characteristics camptothecin concentrations lower than l /IM. of camptothecin-induced DNA lesions in a mammalian cell line When the SSB data (Fig. 9) are considered in light of these have been clarified. High frequencies of DNA strand breaks are results, it can be concluded that the plateau observed in SSB produced (comparable to the most active inhibitors of topoi dose response is an artifact resulting from an increasing loss of somerase II), and they appear to be completely protein linked. 5020

This finding, together with the parallel behavior of camptothe- in experiments where camptothecin is used to map topoisom cin-induced DNA lesions in purified systems and in cells, erase I sites of action in mammalian genes. In addition, in the supports the hypothesis that the cellular lesions are mediated case of alkaline elution experiments, the use of internal standard entirely by topoisomerase I. cells should be either omitted or done by adding them imme diately prior to SDS lysis, in order to avoid an altered elution profile of these cells in response to their exposure to campto DISCUSSION thecin, even at 0Â°C. The demonstration that camptothecin is a potent inhibitor In summary, the protein linkage of DNA breaks produced by of purified mammalian topoisomerase I has resulted in renewed camptothecin and their similar sensitivity to detergents and to interest in the development of camptothecin analogs as ani ilu temperature in cells, in isolated nuclei, and with purified topo mor agents (3) and has also led to the use of camptothecin to isomerase I strongly suggest that inhibition of topoisomerase I localize the sites of topoisomerase I action in eukaryotic genes by stabilization of enzyme-DNA complexes is the unique mech (10-13). However, although there is good evidence that camp anism of production of DNA breaks by camptothecin in mam tothecin inhibits topoisomerase I in mammalian cells (26, 31- malian cells. In addition, the topoisomerase I-DNA complexes 33), previous work suggested that camptothecin-induced DNA that are stabilized by camptothecin appear to be less stable to mild detergents and after drug removal at 0Â°Cthan the topoi breaks in cells are not entirely attributable to topoisomerase I inhibition (14, 15). somerase II-DNA complexes that are stabilized by topoisom The present study demonstrates for the first time that camp erase II inhibitors such as m-AMSA. tothecin produces in mammalian cells approximately equal frequencies of SSB and DPC and that essentially all the DNA REFERENCES fragments generated by the breaks are protein linked. Such an observation is consistent with the possibility that one terminus 1. Wall. M. E., Wani, M. C, Cook, C. E., Palmer, K. H.. McPhail, A. T., and of each DNA single-strand break produced by the drug is tightly Sim, G. A. Plant antitumor agents. I. 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5022 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. Protein-linked DNA Strand Breaks Induced in Mammalian Cells by Camptothecin, an Inhibitor of Topoisomerase I

Joseph M. Covey, Christine Jaxel, Kurt W. Kohn, et al.

Cancer Res 1989;49:5016-5022.

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