(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 :!)!•(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 4°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 0°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 ///»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 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. CAMPTOTHECIN-INDUCED DNA STRAND BREAKS (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 (/•„)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<(ï/nil[•'!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%.