[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-íil 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 dishes (NUNCLONE) with 0.04 tiC\/ with bleomycin (Fig. ID), which inhibits neither topoisomerase ml [14C]thymidine and incubated for 20 h at 37°Cin 5% CO2. At the I nor topoisomerase II. Synergy was observed with TNF con end of the labeling, medium was removed and the monolayer was centrations between 10 and 1000 units/ml. However, since at reincubated with fresh medium for 1 h. Various drugs were then added the high concentration, TNF alone exhibited 15-20% cytotox at appropriate concentrations into culture with or without TNF (1000 icity in the 24-h assay, further experiments of drug synergy in units/ml) and incubation was continued for 1 h. After drug treatment, cells were washed once with PBS (4°C)and then harvested by scraping into PBS (4°C).To these cells were added equal numbers of 3H-labeled U937 cells that had been irradiated with 300 rads in a Gamma cell 40 cesium source (Atomic Energy of Canada, Ltd.) (dose rate, 134 rads/ min). These cells served as internal standards for the elution assay (22). The mixtures of cells were deposited onto polycarbonate membrane filters (2 ßmpore diameter; Nucleopore Corp., Pleasanton, CA) and lysed with a solution containing 0.1 M glycine-0.025 M disodium EDTA-2% sodium dodecyl sulfate, pH 10. with 0.5 mg/ml proteinase K. Elution was carried out with tetrapropylammonium hydroxide- EDTA-0.1% sodium dodecyl sulfate, pH 12.1, at a flow rate of 0.04 ml/min. Fractions were collected every 3 h for a total of 18 h. Single- strand break frequencies were calculated and results were expressed as "rad equivalents" of DNA strand breakage, as described by Kohn et al. (22). Preparation of Nuclear Extracts from Tumor Cells. L929 cells (0.7 x IO7cells) were seeded into 80-cnr flasks and monolayers were estab lished by overnight culture at 37°Cin 5% CO2. Cells were treated with i.o 1000 units/ml TNF for various incubation times and harvested by scraping. Nuclei were prepared by swelling the cells in 1 ml hypotonie buffer (5 mM KPO4, pH 7.0/2 mM MgCl2/l mM phenylmethylsulfonyl fluoride/1 mM 2-mercaptoethanol/0.1 mM EDTA) for 30 min at 4°C and then disrupted by 30 strokes of a Dounce homogenizer. Nuclei were collected by centrifugaron (200 x g, 10 min) and resuspended at 3 x IO7 nuclei/ml in isotonic buffer (5 mM KPO4, pH 7.5/100 HIM NaCl/10 mM 2-mercaptoethanol/0.5 mM phenylmethylsulfonyl fluo ride). NaCl (5 M) was added to a final concentration of 0.35 M (23, 24) and the nuclei were vortexed gently, incubated on ice for 30 min, and sedimented (1000 x g for 10 min). The supernatant was assayed for topoisomerase I or topoisomerase II activity. Determination of Topoisomerase Activity. Topoisomerase I activity in nuclear extracts was assayed using pBR322 DNA as described by Hsiang et al. (16). Five-¿ilportions of diluted extract were mixed with 5 M' of supercoiled pBR322 DNA. The reactions were allowed to io L proceed for 30 min at 37°C,after which the DNA was electrophoresed 1.0 Z.O 0 0.5 1.0 in 1.0% agarose gels in tris borate buffer. DNA was visualized by DRUG CONCENTRATION staining with ethidium bromide after electrophoresis. One unit of Fig. 1. Effect of TNF on the survival of cells treated with chemotherapeutic topoisomerase I activity was defined as the amount that produced drugs. L929 cell monolayers were treated with various concentrations of (A) observable relaxation of 100 ng of the supercoiled DNA substrate. camptothecin, (B) amsacrine. (C) teniposide, or (D) bleomycin in the presence Specific activities were estimated by dividing the activity (units/ml) by (•)or absence (O) of 100 units/ml TNF for 20 h. Cell survival curves were determined by the crystal violet dye exclusion assay. The survival of cells treated the protein concentration of the extracts, determined by the method of with TNF (100 units/ml) alone was 90.8% (A), 92.5% (B). 95.1% (C). and 94.8% Bradford (25). (O). Drug concentrations are expressed as fi\i (A-C) or units/ml (O). Bars, SEM. 2637

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. TNF AND DNA TOPOISOMERASES the cytotoxicity assay were carried out using 100 units/ml of in the DNA strand breakage assays, except for the omission of TNF. Although in these experiments synergy was detected camptothecin and topoisomerase II inhibitors that trap the following a 24-h incubation with TNF and drugs, similar effects reaction intermediates, TNF treatment induced rapid and tran were observed when 1, 2, or 4 h of incubation with TNF or sient increases in extractable topoisomerase I and II activities drugs was followed by 12-18 h incubation in medium alone. (Fig. 3). Maximal activity was achieved 2-5 min after TNF Moreover, results obtained from the 24-h dye exclusion assay, treatment and by 30-60 min it was equal to or lower than the which does not differentiate between cytotoxic and cytostatic activity extracted from nuclei of untreated cells. effects of drugs, are in agreement with data obtained in the 5lCr Kinetics of TNF-induced Potentiation of DNA Strand Break release cytotoxicity assay (8). age and Cell Killing by Topoisomerase Inhibitors. To ascertain Potentiation by TNF of DNA Strand Breakage Induced by whether the transient nature of the enhancement of extractable Inhibitors of Topoisomerase I or Topoisomerase II. TNF (1000 topoisomerase activity induced by TNF is reflected in the units/ml) incubated for 60 min with L929 cells produced no potentiation of DNA strand breakage induced by topoisomerase DNA strand breakage (Fig. 2). However, when incubated for inhibitors, cells were treated with TNF for 0, 0.5, or 3 h before 60 min in the presence of either camptothecin (Fig. 2, left) or the addition of camptothecin or amsacrine. The effect on strand amsacrine (Fig. 2, right), TNF increased numbers of DNA breakage was evaluated after an additional coincubation of 1 h. single strand breaks generated by these two topoisomerase As demonstrated in Table 2, TNF-mediated potentiation of inhibitors. Similarly, simultaneous treatments of cells with camptothecin- or VP16-induced strand breakage was achieved TNF and other topoisomerase inhibitors consistently enhanced only when TNF was added simultaneously with the topoisom DNA strand breakage (Table 1). erase inhibitors. Preincubation of the cells with TNF for a Effect of TNF Treatment on Extractable Topoisomerase Ac period as short as 30 min abolished the synergy with topoisom tivity. Topoisomerase I and II activities can be extracted from erase inhibitors. isolated nuclei by incubating them at 4°Cin buffer containing To further determine whether TNF-induced transient eleva 0.35 M NaCl (23, 24). Under conditions identical to those used tion of extractable topoisomerase activity and potentiation of DNA strand breakage were kinetically associated with the po tentiation of cytotoxicity induced by topoisomerase inhibitors, the effect of pretreatment with TNF on topoisomerase inhibi tor-mediated cytotoxicity was investigated. L929 cells were incubated for 4 h either with TNF (group A) or with topoisom erase inhibitors (camptothecin or amsacrine) (group B). After this initial treatment, the supernatants were carefully aspirated and without further washings of the cells, medium containing

1.0 O.I 1.0 O.I I i FRACTION OF C'H 3 - RADIOACTIVITY RETAINED 200 Fig. 2. Effect of addition of TNF on DNA strand breakage induced by in inhibitors of topoisomerase I or topoisomerase II. 14C-labeled L929 cell mono- layers were treated with medium only, 10 MMcamptothecin (left) or 0.4 MM amsacrine (right) in the presence or absence of 1000 units/ml TNF for 1 h. Cells were harvested and DNA strand breakage was assayed as described in "Materials and Methods." Data are representative of more than three separate experiments: Left: medium control (O). TNF (•),camptothecin (A), camptothecin plus TNF ü (A); right: medium control (O), TNF (•).amsacrine (A), amsacrine plus TNF < 60 (A). u Table 1 Effect of TNF on protein-concealed DNA strana breaks induced by lu topoisomerase I or II inhibitors11 o.

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. TNF AND DNA TOPOISOMERASES

Table 2 Effect of order of TNF addition on DNA strand breakage induced by topoisomerase I or 11inhibitors" however, is not shared by other cytotoxic drugs that do not act by topoisomerase inhibition. Despite their differences in mech TNF treatment before Drug addition DNA strand breaks Experiment drug addition (1 h) (rad equivalents) anism and cellular functions, topoisomerase I and II both catalyze the formation of reaction intermediates that can be 1 None None CPT + TNF 1095(54)' trapped by inhibitors as cleavable complexes. These complexes h2 TNF, 0.5 CPTCPT 728(3)364 are likely lethal to cells if they are not reversed. Therefore, the None effect of TNF on DNA strand breakage induced by topoisom None CPT + TNF 773(112) erase inhibitors was evaluated. The results of the elution exper h3 TNF, 3 CPTVP16 403(11)497 iments clearly demonstrate TNF-induced potentiation of cleav

None able complex formation generated following treatment with None VP16 + TNF 622 (25) camptothecin or etoposide and teniposide. The increase in h4 TNF, 0.5 VP16VP16 497(0)676 DNA strand breaks was not caused by increased drug accumu None lation after TNF treatment (data not shown). The mechanism NoneCPT* VP16 + TNF709 952(41) and kinetics of the enhanced etoposide- or camptothecin-in- " Tumor cells were treated with TNF (1000 units/ml) for 0, 0.5, or 3 h prior duced DNA strand breaks are consistent with the transient to the addition to the incubation medium of camptothecin (10 UM)or etoposide (10 MM)and further incubation for l h. L929 cells treated with TNF for 4 h did increases in extractable topoisomerase I and II activities de not sustain DNA strand breakage. tected in TNF-treated L929 cells. These results suggest that * CPT, camptothecin; VP16, etoposide. ' Numbers in parentheses, percentage increase in DNA strand breaks over the TNF increases the number of topoisomerase I and II molecules number produced by 1 h incubation with the topoisomerase inhibitor alone. which are active and thus are trapped and detected as strand breaks in the elution assays. This would increase cellular sen Table 3 Effect of sequence of drug addition on cytotoxicity against L929 cells" sitivity to killing by inhibitors such as camptothecin, amsacrine, First treatment treatment or teniposide because the ability of these inhibitors to form (4h)MediumMediumTNFCamptothecinMediumTNFAmsacrineSecond<4h)TNFCamptothecinCamptothecinTNFAmsacrineAmsacrineTNF%ofcytotoxicity5.6 toxic lesions is proportional to the number of active topoisom ±2.2"21.8 erase molecules per cell. ±0.827.7 The transient nature of the activation of extracted topo- ±3.440.4 ±0.536.3 isomerases I or II by TNF may reflect either real activation and deactivation of the enzymes or an increase followed by a de ±0.945.6 ±3.060.3 crease in the extractability of the enzymes, possibly because ±0.3 they bind more tightly to chromatin in the activated state. °L929 cells were treated with medium containing indicated agents for 4 h These observations are consistent with the findings that camp (first treatment) and then supernatants were replaced with medium containing tothecin- or VM26-induced DNA strand breakage and cell indicated agents for another 4 h (second treatment). After 8 h, cells were incubated with fresh medium for an additional 12 h. Camptothecin. amsacrine. and TNF cytotoxicity were not enhanced by TNF if the inhibitors were were used at concentrations of 2 /IM, 2.5 /IM, and 100 units/ml, respectively. not present before or immediately after the addition of TNF to * Mean ±SD of three determinations. the cells. Thus, kinetically, a cause-effect relationship may exist between TNF-induced activation of topoisomerases and the topoisomerase inhibitors (group A) or TNF (group B) was enhanced DNA damage and cell killing by topoisomerase in added and all cultures were incubated for an additional 4 h. hibitors. Control cultures were treated with camptothecin, amsacrine, or Several possible mechanisms for the observed change in the TNF for 4 h. Supernatants were removed from all cultures and activity of extractable topoisomerase following TNF treatment replaced with fresh drug-free medium and incubation was con may be suggested. It is well known that TNF binds to specific tinued at 37°Cfor an additional 12 h. The results in Table 3 cellular receptors (27, 28) and induces eicosanoid synthesis demonstrate that augmentation of cytotoxicity was found only (29-31) and c-fos and c-myc gene expression (32) in certain cell when L929 cells were treated with topoisomerase inhibitors lines. Also, it has been demonstrated that TNF activates phos- prior to addition of TNF. In contrast, no synergy was detected pholipase A2-activating protein in bovine endothelial cells 3- when cells were incubated with TNF for 4 h before treatment 10 min after its addition to the cultures (33). The expression of with camptothecin or amsacrine. Thus, exposure of the cells to this protein is effected by an inositol phosphate-linked cellular TNF alone for periods longer than 10 min, when the TNF- signaling pathway in which the production of diacylglycerol induced increase in topoisomerase activity had reversed to and the mobilization of calcium rapidly activate protein kinase control levels, resulted in the abrogation of the TNF-mediated C (34). Protein kinase C can phosphorylate topoisomerases, potentiation of DNA damage and cytotoxicity induced by to thereby increasing their activity (35-37). Thus, TNF has the poisomerase inhibitors. potential to activate topoisomerases in the experiments re ported here via protein kinase C. Reversal of the activation could result from feedback or desensitization which are within DISCUSSION the capability of a protein-phosphorylating enzyme such as This study demonstrates the augmentation of tumor cell protein kinase C. An alternate mechanism is suggested by a killing by treatment with a combination of TNF and topoisom recent report that TNF induces a rapid (3-5-min) increase in erase I and II inhibitors and provides experimental evidence in cyclic AMP and cyclic AMP-dependent protein kinase in hu support of possible molecular mechanisms that may be respon man FS-4 fibroblast cells. This increase was also transient (38). sible for this synergistic activity. Recent studies have demon Thus, it is possible in our experiments that TNF-receptor strated synergy between topoisomerase II inhibitors and TNF binding could have rapidly and transiently activated topo (1) or natural cytotoxic cell-mediated cytotoxicity in killing isomerases via cAMP-dependent cellular signaling pathways. L929 cells (8). Here we extended this observation by demon Irrespective of the exact mechanism, the transient nature of the strating synergy between TNF and the topoisomerase I inhibi increase in extractable topoisomerase activity by TNF was tor, camptothecin, in L929 cell cytotoxicity. This property, reflected in the ability of TNF to potentiate cell killing induced 2639

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. TNF AND DNA TOPOISOMERASES protein-linked DNA breaks via mammalian DNA topoisomerase I. J. Biol. by topoisomerase inhibitors (Fig. 3). This result provides a link Chem., 27: 14873-14878, 1986. between the observed biochemical effect of TNF on topoiso- 17. Tewey. K. M.. Chen, G. L., Nelson, E. M., and Liu, L. F. Intercalative merases and its cytotoxic effect on the cultured cells. antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. J. Biol. Chem., 259: 9182-9187. 1984. Recent reports show that TNF enhanced cytotoxic effects of 18. Ross. W. E.. Glaubiger. D. L.. and Kohn. K. W. Protein-associated DNA topoisomerase II inhibitors in vivo (2) and demonstrated syn- breaks in cells treated with Adriamycin or ellipticine. Biochim. Biophys. ergistic cytotoxicity between natural cytotoxic cells and topo Acta, 519: 23-30, 1978. 19. Hsiang. Y-H., and Liu. L. F. Identification of mammalian DNA topoisom isomerase I or II inhibitors to the killing of L929 cells. In the erase I as an intracellular target of the anticancer drug camptothecin. Cancer latter study, tumor cell killing was due to TNF secreted from Res., 48: 1722-1726. 1988. 20. Yang, L.. Rowe. T. C., Nelson, E. M., and Liu, L. F. In vivo mapping of natural cytotoxic cells (8). These results suggest that the aug DNA topoisomerase-specific cleavage sites on SV40 chromatin. Cell. 41: mentation of cytotoxicity produced by topoisomerase inhibitors 427-432. 1985. can be enhanced not only by exogenous TNF but also by TNF 21. Yang, L.. Rowe. T. C., and Liu, L. F. Identification of topoisomerase II as the intracellular target of antitumor epipodophyllotoxins in simian virus 40- that is secreted by certain immune cytotoxic cells. These results infected monkey cells. Cancer Res., 45: 5872-5876. 1985. may have relevance to the selection of immunochemotherapeu- 22. Kohn. K. W., Ewig, R. A. G.. Erickson, L. C.. and Zwelling, L. A. Measure tic protocols for cancer treatment. ments of strand breaks and cross-links by alkaline elution. In: E. C. Friedberg and P. C. Hanawalt (eds.), DNA Repair: A Laboratory Manual of Research Techniques, pp. 379-406. New York: Marcel Dekker. Inc., 1981. 23. Liu, L. F.. and Miller. K. G. Eukaryotic DNA topoisomerases: two forms of REFERENCES type I DNA topoisomerase from HeLa cell nuclei. Proc. Nati. Acad. Sci. USA. 78: 3487-3491, 1981. 24. Miller, K. G.. Liu, L. F., and Englund, P. T. A homogeneous type II DNA 1. Alexander. R. B.. Nelson. W. G., and CofTey. D. S. Syncrgistic enhancement topoisomerase from HeLa cell nuclei. J. Biol. Chem.. 256:9334-9339. 1981. by tumor necrosis factor of in vitro cytotoxicity from chemotherapeutic drugs targeted at DNA topoisomerase II. Cancer Res.. 47: 2403-2406. 1987. 25. Bradford, M. A. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing principles of protein-dye binding. 2. Alexander. R. B., Isaacs. J. T., and Coffey, D. S. Tumor necrosis factor Anal. Biochem.. 72:248-254. 1976. enhances the in vitro and in vivo efficacy of chemotherapeutic drugs targeted 26. Tan. K. B.. Mattern. M. R., Boyce, R. A., and Schein, P. S. Elevated DNA at DNA topoisomerase II in the treatment of murine bladder cancer. J. Urol.. topoisomerase II activity in nitrogen mustard-resistant human cells. Proc. 138: 427-429, 1987. Nati. Acad. Sci. USA. 84: 7668-7671, 1987. 3. Watanabe. N., Niitsu. Y.. Yamaguchi. N., Ohtsuka. Y., Sone, H.. Neda. H.. 27. Baglioni. C.. McCandless, S., Tavernnier, J., and Fiers, W. Binding of human Maeda. M., and Urushizaki. I. Synergistic cytotoxicity of recombinant human tumor necrosis factor to high affinity receptors on HeLa and lymphoblastoid TNF and various anti-cancer drugs. Immunopharmacol. Immunotoxicol., cells sensitive to growth inhibition. J. Biol. Chem., 260: 13395-13397. 1985. 10: 117-127. 1988. 28. Hass. P. E., Hotchkiss. S., Mohler. M., and Aggarwal, B. B. Characterization 4. Flick. D. A., and Gifford. G. E. Comparison of in vitro cell cytotoxic assay of specific high affinity receptors for human tumor necrosis factor on mouse for tumor necrosis factor. J. Immunol. Methods. 6K: 167-175. 1984. fibroblasts. J. Biol. Chem., 260: 12214-12218. 1985. 5. Ziegler-Heitbrock. H. W. L., and Riethmuller, G. A rapid assay for cytotox 29. Backwich, P. R.. Chensue. S. W., Larrick, J. W., and Kunkel. S. L. Tumor icity of unstimulated human monocytes. J. Nati. Cancer Inst., 72: 23-29. necrosis factor stimulates interleukin-1 and prostaglandin 1 . production in 1984. resting macrophages. Biochem. Biophys. Res. Commun., /.?6:94-101, 1986. 6. Coletta, F.. Peri, G., Villa. A., and Mantovani, A. A rapid killing of actino- 30. Dayer. J-M., Beutler. B., and Cerami. A. Cachectin/tumor necrosis factor mycin D-treated cells by human mononuclear cells. 1. Effectors belong to the stimulates collagenase and prostaglandin I production by human synovial monocyte-macrophage linkage. J. Immunol., 132: 936-944, 1984. cells and dermal fibroblasts. J. Exp. Med., ¡62:2163-2168, 1985. 7. Coletta. F.. Bersani. L., Lazzarin. A., Poli, G., and Mantovani. A. Rapid 31. Nawroth. P. P.. and Stern. D. M. Modulation of endothelial cells' hemostatic killing of actinomycin D-treated tumor cells by human monocytes. II. Cyto properties by tumor necrosis factor. J. Exp. Med., 163: 740-745, 1986. toxicity is dependent on secretion of reactive oxygen intermediates and is 32. Lin, J-X., and Vilcek, J. Tumor necrosis factor and interleukin-1 cause a suppressed by protease inhibitors. J. Immunol.. 134: 3524-3531. 1985. rapid and transient stimulation of c-fos and c-myc mRNA levels in human 8. Utsugi, T., Demuth. S.. and Manna, N. Synergistic antitumor effects of fibroblasts. J. Biol. Chem.. 262: 11908-11911. 1987. topoisomerase inhibitors and natural cell-mediated cytotoxicity. Cancer Res., 33. Clark. M.. Chen. M.-J., Crooke, S. T.. and Bomalaski. S. Tumour necrosis 49: 1429-1433. 1989. factor (cachectin) induces phospholipase A2 activity and synthesis of a phos- 9. Wang, J. C. DNA topoisomerases. Annu. Rev. Biochem., 54:665-679, 1985. pholipase A2-activating protein in endothelial cells. Biochem. J.. 250: 125- 10. Liu, L. F. DNA topoisomerases—enzymes that catalyze the breaking and 132, 1988. rejoining of DNA. CRCCrit. Rev. Biochem.. IS: 1-24, 1983. 34. Crooke. S. T.. Mong, S., Clark, M.. Sarau. H., Wang, A., Vegesna. R., 11. Glisson, B. S.. and Ross, W. E. DNA topoisomerase II: a primer on the Winkler, J.. Balcarek. J.. and Bennett. C. F. LTD4 receptor and signal enzyme and its unique role as a multidrug target in cancer chemotherapy. transduction processes. In: G. Poste and S. T. Crooke (eds.). Cellular and Pharmacol. Ther.. 32: 89-106. 1987. Molecular Aspects of Inflammation, pp. 321-333. New York: Plenum Pub 12. Tewey. K. M., Rowe. T. C., Yang, L.. Halligan. B. D., and Liu. L. F. lishing Corp.. 1988. Adriamycin-induced DNA damage mediated by mammalian DNA topoisom 35. Durban. E.. Mills, J. S.. Roll. D.. and Busch, H. Phosphorylation of purified erase II. Science (Wash. DC), 226: 466-478. Õ984. Novikoff hepatoma topoisomerase I. Biochem. Biophys. Res. Commun., Ill: 13. Ross, W.. Rowe, T.. Glisson, B.. Yalowich. J.. and Liu, L. Role of topoisom 897-905. 1983. erase II in mediating epipodophyllotoxin-induced DNA cleavage. Cancer 36. Kaiserman, H. B.. Ingebritsen. T. S.. and Benbow, R. M. Regulation of Res., 44: 5857-5860, 1984. Xenopus laeris DNA topoisomerase I activity by phosphorylation in vitro. 14. Nelson, E. M., Tewey, K. M., and Liu. L. Mechanism of antitumor drug Biochemistry. 27:3216-3222. 1988. action: poisoning of mammalian DNA topoisomerase II on DNA by 4'-(9- 37. Ackerman, P., Glover, C. V. C., and Osheroff, N. Phosphorylation of DNA acridinylamino)methanesu!fon-m-anisidide. Proc. Nati. Acad. Sci. USA. 81: topoisomerase II by casein kinase II: modulation of eukaryotic topoisomerase 1361-1365. 1984. II activity in vitro. Proc. Nati. Aead. Sci. USA, 82: 3164-3168. 1985. 15. Rowe. T. C., Chen, G. C.. Hsiang. Y., and Liu. L. DNA damage by antitumor 38. Zhang, Y., Lin, J-X., Yip, Y. K., and Vilcek, J. Enhancement of cAMP levels acridines mediated by mammalian DNA topoisomerase II. Cancer Res.. 46: and of protein kinase activity by tumor necrosis factor and interleukin 1 in 2021-2026. 1986. human fibroblasts: role of the induction of interleukin 6. Proc. Nati. Acad. 16. Hsiang. Y-H., Hertzbcrg, R.. Hecht. S., and Liu, L. F. Camptothecin induces Sci. USA. 85: 6802-6805. 1988.

2640

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. 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, et al.

Cancer Res 1990;50:2636-2640.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/50/9/2636

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/50/9/2636. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research.