[CANCER RESEARCH 49, 6202-6207, November 15, 1989] Altered Formation of DNA in Human Cells Treated with Inhibitors of DNA Topoisomerase II (Etoposide and Teniposide)1 Ulf Lönn,2Sigrid Lönn,Urban Nylen, and Gerard Winblad Radiumhemmet, Karolinska Hospital [U. L., S. L., U. N., G. W.], and Department of Histology, Karolinska Institute! [U. L., S. L.], P.O. Box 60500, S-I04 01 Stockholm, Sweden ABSTRACT of this, it is of interest to examine the influence of the enzyme on a defined stage in DNA synthesis. We have investigated the importance of DNA topoisomerase II for the To analyze the effect on DNA synthesis of inhibition of DNA formation of mammalian DNA replication intermediates. Treatment with topoisomerase II, we treated cells with etoposide (or tenipo the DNA topoisomerase II inhibitor etoposide (teniposide) prevents the formation of large intermediates, such as 10-kilobase DNA, but allows side), which is believed to be a specific inhibitor of DNA the formation of small intermediates, i.e.. Okazaki fragments. In un topoisomerase II (13, 14). We find that the drug treatment treated cells, there is a distinct stage in which the 10-kilobase DNA prevents the appearance of the large 10-kilobase DNA inter intermediates are joined before the appearance of mature eliminatili. We mediate and abolishes the appearance of the post-elongation find that pretreatment with etoposide (teniposide) prevents the appear stage. ance of this stage. When the protocol is reversed and the cells contain labeled 10-kilobase DNA before exposure to the drugs, one can detect MATERIALS AND METHODS the stage. Cells, Culture Medium, and Labeling with |3H|Thymidine. A human melanoma cell line (CRL 1424), obtained from Flow Laboratories, Irvine, United Kingdom, was grown as monolayers at 37°Cin 5% CO2 INTRODUCTION in air. The culture medium was Eagle's minimal essential medium with Mammalian DNA synthesis occurs by a semicontinuous bi Earle's salts, containing 2 ITIML-glutamine, 10% fetal calf serum, and directional mechanism. Small replication intermediates are antibiotics. joined into larger intermediates, and finally adjacent replicon- For experiments the cells were seeded in small culture dishes (35 x 10 mm) containing 3 ml of medium. Twenty-four h later, the cells were sized DNA fragments are joined. Our knowledge of these events incubated with 50 pCi of [3H]thymidine (22 Ci/mmol; Amersham, Inc., is rather incomplete. Recently we have shown that the primary Amersham, United Kingdom) for the desired length of time. To obtain replication intermediates, Okazaki fragments, form large inter prelabeled DNA, the cells were incubated for 24 h in ['Hjthymidine- mediates (10-kilobase DNA) and that, after the joining of the containing medium and then for another 24 h in medium without 10-kilobase DNA intermediates, one can detect a distinct post- synthetic DNA stage (called "the post-elongation stage") before thymidine. Etoposide and teniposide were obtained from a local pharmacy. the appearance of mature chromatin DNA (1). Cell Lysis. The incubation medium was drained from the culture We know some of the enzymes that are involved in the dish, and the cells were rinsed twice with cold phosphate-buffered saline. Cell lysis was performed in the dark at 0°Cbythe addition of 2.25 ml formation of Okazaki fragments, e.g., DNA polymerase. We know also that poly(ADP-ribose)ylation is involved in the join of 0.03 M NaOH (pH 12.1). After 30 min the solution was neutralized ing processes. When ribosylation is inhibited, large replication with 0.9 ml of 0.067 M HC1/0.02 M NaH2PO4. For a more detailed intermediates accumulate (2), and the characteristics of the description, see Refs. 1 and 15. The sample was then either digested "post-elongation stage" are altered (3). with nuclease S, (see below) or immediately made 0.5% with regard to SDS.3 The topologica! constraints involved in DNA replication are Digestion with Nuclease Si. Immediately after the neutralization of now becoming known. Recent data have shown that the enzyme the dilute alkali used to lyse the cells, 300 M'of 300 mM sodium acetate DNA topoisomerase II is a structural protein of interphase (pH 4.6)-0.5 mM zinc acetate-750 mM NaCl were added. Two-hundred chromatin (4) and mitotic chromosomes (5, 6). There is evi lU/ml of nuclease S, (Sigma Chemical Co., St. Louis, MO) were then dence to implicate the enzyme in DNA replication and segre added, and the mixture was incubated for 30 min at 37°C.Thedigestion gation of chromosomal material at the end of the replication was stopped by making the solution 1% SDS-0.02 M EDTA. as well as at mitosis (7-9). Agarose Gel Electrophoresis. Agarose gels (0.75%) were made as During DNA synthesis unwinding of DNA helices must earlier described (1). The labeled DNA was separated in the gels using an LKB Multiplier electrophoretic system. The gels were cut into 1- occur; conditions that favor or prevent unwinding should facil mm-thick slices, and the radioactivity was measured in a toluene-based itate or inhibit DNA replication. An important condition is the scintillation fluid containing 3% Soluene 100, using a Packard scintil superhelical tension of DNA which is relaxed by the enzyme lation counter. DNA topoisomerase I and II (10, 11). The enzyme action has Treatment of Nuclei in Salt Solutions. Isolated nuclei were suspended been studied extensively in vitro, but only recently one has been in buffer (0.25 M sucrose-50 mM Tris-HCl (pH 7.5)-25 mM KC1-1 mM able to analyze the importance of the enzyme in vivo. CaCl2-l mM MgCl2). KC1 was added to a final concentration of 0.3 M It has been proposed that DNA topoisomerase II is a specific or 0.6 M, and the nuclei were incubated at 37°Cin order to reduce the marker for cell proliferation (12). The appearance of the enzyme nucleosome repeat length (16, 17). In nuclei incubated in 0.3 M KC1 parallels the start of DNA synthesis, which led the authors to the repeat length is 160 to 170 base pairs and, in nuclei incubated in suggest that the enzyme is involved in DNA synthesis. In view 0.6 M KC1, it is 140 to 145 base pairs. The nucleosome repeat length in nuclei incubated in sucrose-buffer alone is 190 to 195 base pairs. Received 1/20/89; revised 4/21/89, 7/20/89; accepted 8/10/89. KC1/SDS Precipitation of Protein-DNA Complexes. Nuclei were lysed The costs of publication of this article were defrayed in part by the payment in 2% SDS-10 mM EDTA-10 mM Tris (pH 8.0). The lysates were then of page charges. This article must therefore be hereby marked advertisement in subjected to the KC1/SDS assay, the same day as the lysate was accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by grants from the Swedish Cancer Society and the obtained. The precipitation procedure, using KC1 and SDS, was as Karolinska Institute!. described in Ref. 18. 2To whom requests for reprints should be addressed, at Radiumhemmet. Karolinska Hospital. P.O. Box 60500, S-104 01 Stockholm, Sweden. 3The abbreviationusedis:SDS. sodium dodecylsulfate. 6202 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1989 American Association for Cancer Research. ALTERED FORMATION OF DNA IN ETOPOSIDE (TENIPOSIDE)-TREATED CELLS RESULTS min are pulsed with [3H]thymidine for 45 s at the beginning or at the end of treatment (Fig. IA). The gel electrophoretic To analyze the importance of different enzymes for the separation shows high-molecular-weight DNA and DNA mi various stages of in vivo DNA synthesis, one needs to be able grating as Okazaki fragments as well as another population to distinguish between different replication intermediates. This which is smaller than 10 kilobases. One can detect a small is difficult because of the large size of the mammalian genome. amount of 10-kilobase DNA intermediates. The results are To partly overcome problems resulting from the large size of mammalian genome, we have developed a procedure of cell similar irrespective of whether the pulse labeling was performed lysis in dilute alkali at 0°Cto partly denature the DNA. During at the beginning or at the end of treatment. In cells pulsed with [3H]thymidine during the last 45 s of a the alkaline treatment, macromolecules are removed from the 30-min drug treatment, one can detect essentially the same DNA, and the base pair structure is disrupted. However, the picture as in cells treated with drug for 5 min, i.e., high- DNA strands cannot separate before enough time has elapsed molecular-weight DNA, Okazaki fragments, and the new pop to allow unwinding of the DNA. The unwinding is initiated at gaps (and/or alkali-labile regions) in the DNA present, e.g., at ulation of fragments. However, now one cannot detect 10- the DNA replication forks (1, 15). The amount of DNA that kilobase DNA intermediates (Fig. IB). may be unwound at each gap has been estimated as 20 kilobases. Treatment with Teniposide. Teniposide is an analogue to When the number of gaps is increased by, e.g., X-irradiation, a etoposide which similarly inhibits DNA topoisomerase II (13, higher amount of DNA is denatured. 14). In parallel to the experiments described above for etopo When the solution is neutralized, the high-molecular-weight side, we have performed incubations with teniposide according DNA is renatured and forms double-stranded DNA. DNA to the same protocols. Fig. 2 shows that teniposide prevents replication intermediates smaller than 20 kilobases, which are the appearance of the 10-kilobase DNA replication intermedi released from the DNA during the unwinding, remain in solu ate.