Potentiation of Topoisomerase Inhibitor-Induced DNA Strand Breakage and Cytotoxicity by Tumor Necrosis Factor: Enhancement of To
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[CANCER RESEARCH 50. 2636-2640, May I. 1990] Potentiation of Topoisomerase Inhibitor-induced DNA Strand Breakage and Cytotoxicity by Tumor Necrosis Factor: Enhancement of Topoisomerase Activity as a Mechanism of Potentiation Teruhiro Utsugi, Michael R. Mattern, Christopher K. Mirabelli, and Nabil Hanna1 Departments of Immunology [T. I'., ,V. H.¡and Molecular Pharmacology [M. R. M., C. K. M.¡,Smith Kline & French Laboratories, King of Prussia, Pennsylvania 19406- 0939 ABSTRACT onstrated that treatment of target cells with topoisomerase I or II inhibitors enhanced their killing by natural cytotoxic cell- A combination of tumor necrosis factor (INI) and the topoisomerase mediated cytotoxicity (8). I inhibitor, camptothecin, or the topoisomerase II inhibitors, teniposide and amsacrine, produced dose-dependent synergistic cytotoxicity against Topoisomerases are nuclear enzymes which catalyze the for the murine L929 fibrosarcoma cells. Similar synergy was not observed mation of various topological isomers of DNA by transiently breaking and rejoining DNA (9-12). Topoisomerase I catalyzes with a combination of TNF and bleomycin. To define the role of TNF in the augmentation of tumor cell killing by topoisomerase I or II inhibitors, single strand breakage and strand passage independent of ATP, the effect of TNF on the production of enzyme-linked DNA strand breaks whereas topoisomerase II catalyzes double strand DNA break induced in cells by topoisomerase inhibitors was investigated. L929 cells age and strand passage in the presence of ATP. Topoisomerase incubated for l h with the topoisomerase inhibitors contained protein- inhibitors are believed to induce cytotoxicity by stabilizing linked strand breaks. In contrast, INI alone did not induce DNA strand covalent complexes of enzyme and strand-cleaved DNA, which breakage. However, when cells were incubated simultaneously with TNF are normally transient intermediates of the topoisomerase re and camptothecin, amsacrine, Adriamycin, actinomycin D, teniposide, or action mechanism (12-15). The antitumor agent camptothecin etoposide, increased numbers of strand breaks were produced. Preincu- is the only known inhibitor of topoisomerase I that works by bation of the cells with INI for 30 min or 3 h before the addition of camptothecin or etoposide resulted in no more strand breaks than that this mechanism (16). A large number of antitumor agents observed in cells incubated with the drugs alone. TNF treatment of L929 including intercalators such as amsacrine, actinomycin D, and cells produced a rapid and transient increase in specific activity of ellipticine (12, 14, 17) and epipodophyllotoxins (13) such as extractable topoisomerases I and II. These increases were maximum at etoposide and teniposide inhibit topoisomerase II by this mech 2-5 min of IM treatment and by 30 min the activities of extractable anism. In cells, these inhibitors produce strand breaks which enzymes were equal to or less than those detected in extracts from can be assayed by alkaline elution performed in the presence of untreated cell controls. The transient nature of the increase in extractable proteinase (18). The proteins linked covalently to the broken topoisomerase activity may explain the kinetics and significance of the DNA strands have been identified immunologically as topo order of addition of TNF and inhibitors for maximal synergistic activity. isomerase I for camptothecin (19) and topoisomerase II for These data are consistent also with a role for topoisomerase-linked DNA lesions in the TNF-mediated potentiation of killing of L929 cells by intercalators (20) and epipodophyllotoxins (21). topoisomerase inhibitors. We wished to investigate the mechanism by which TNF and topoisomerase inhibitors synergize in killing tumor cells. In particular, we attempted to determine whether TNF increased INTRODUCTION the number of available targets for the topoisomerase inhibitors The availability of recombinant cytokines (TNF,2 interferon, in the cells. Using L929 cells, we first demonstrated synergistic cytotoxicity between TNF and inhibitors of both classes of interleukin 2, and cerebrospinal fluid, etc.) renders combination topoisomerase and then determined that the ability of TNF to therapies involving cytokines and classical chemotherapeutic enhance cell killing by the inhibitors is correlated quantitatively drugs possible. Accordingly, a better understanding of the with its ability to increase topoisomerase I- or II-associated mechanisms of action of cytokines and chemotherapeutic drugs DNA strand breakage as well as to increase the level of extract- will result in the design of more effective protocols involving able topoisomerase catalytic activity. combination therapies. Indeed, in experimental models, com binations between TNF and various chemotherapeutic drugs have been demonstrated to exhibit cytotoxicities against tumor MATERIALS AND METHODS cells (1-4). For example, it is well known that actinomycin D potentiates the killing of several tumor cells by TNF and other Reagents. The chemotherapeutic drugs actinomycin D, Adriamycin, immune effector cells (4-7). However, the mechanism of this and bleomycin were purchased from Sigma Chemical Co., St. Louis, synergy, which may be common to a number of drugs, is MO. Amsacrine, camptothecin, etoposide, and teniposide were kindly unknown. Recent studies have demonstrated that chemothera provided by the Drug Synthesis and Design and Natural Products Branches of the National Cancer Institute, Bethesda, MD. Human peutic drugs that inhibit the nuclear enzyme topoisomerase II recombinant TNF (specific activity, 2 x 10s units/mg protein) was synergize with TNF in tumor cell killing (1, 2). We have provided by J. Chen, Smith Kline & French Laboratories, King of investigated the mechanism of synergy between immune cyto- Prussia, PA. toxic cells and various chemotherapeutic drugs and have dem- Tumor Cells. L929 murine fibrosarcoma cells were maintained in CMEM at 37°Cin a humidified atmosphere of 5% CO2 in air. U937 Rcceived 1/26/89; revised 11/16/89. The costs of publication of this article were defrayed in part by the payment human monoblast cells were maintained in RPMI 1640 supplemented of page charges. This article must therefore be hereby marked advertisement in with 10% fetal bovine serum. Tumor cells were routinely examined and accordance with 18 U.S.C. Section 1734 solely to indicate this fact. found to be free of Mycoplasma contamination. ' To whom correspondence should be addressed. 2The abbreviations used are: TNF. tumor necrosis factor; CMEM, Eagle's Cytotoxicity Assay. The cytotoxicity of L929 cells was determined minimal essential medium supplemented with 10% fetal bovine serum, sodium by the crystal violet dye exclusion assay as described previously (4). pyruvate, essential amino acids, nonessential amino acids. L-glutamine. 2x vita L929 cells were harvested by treatment with 0.25% trypsin-0.02% min solution, penicillin, and streptomycin; PBS, phosphate-buffered saline. EDTA solution and washed once with CMEM. Cells were counted and 2636 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. TNF AND DNA TOPOISOMERASES seeded into 96-well flat bottom plates at a density of 3 x 104/well jp Topoisomerase II activity in nuclear extracts was assayed by the 100 ß\CMEM and monolayers were established by overnight incuba unknotting of P4 phage DNA as described by Tan et al. (26). Five-Ãil tion at 37°Cin 5% CO2. Before assay, medium was discarded and portions of diluted extract were mixed with 5 p\ of knotted P4 DNA. The reactions were allowed to proceed for 30 min at 37°C,after which replaced with 200 /¿Ioffresh medium containing various concentrations of drugs with or without TNF. After 20 h incubation, the cells were the DNA was electrophoresed in 0.7% agarose gels in tris phosphate washed twice with warmed PBS and fixed by methanohacetic acid (3:1) buffer and visualized by ethidium bromide staining. One unit of topoi for 30 s. The monolayers were stained with 0.5% crystal violet in 10% somerase II activity was defined as the amount that removed knots ethanol for 15 min at room temperature. Excess dye was washed off completely from 200 ng of P4 DNA. Specific activities were determined with deionized water and the plates were dried. The amount of dye as described above. associated with the cells was determined after solubilization with 150 Experiments determining topoisomerase activity contained in nu lA of a 10% acetic acid, 20% ethanol solution by measuring absorbance clear extracts were performed at least four times with reproducible by a microelisa reader with a 600 nm filter. The percentage of killing results. was calculated as % of cytotoxicity = I 1 - -1 x 100 RESULTS in which 7" ¡sabsorbance from cells incubated with drugs and C is Synergistic Cell Killing Induced by TNF and Topoisomerase Inhibitors. Enhancement of killing of L929 cells was observed absorbance from cells incubated with medium or TNF alone in drug after combination treatments with 100 units/ml TNF and the synergy experiments. Determination of DNA Strand Breakage. DNA filter elution assays topoisomerase I inhibitor camptothecin (Fig. IA) or the topo was performed as described by Kohn et al. (22). L929 cells were isomerase II inhibitors amsacrine (Fig. IB) or teniposide (Fig. harvested from culture flasks and 4x10* cells in 2 ml CM EM were 1C). In contrast, TNF did not synergize in tumor cell killing seeded into 60- x 15-cm culture