Antagonistic Effect of Aclarubicin on Daunorubicin-Induced Cytotoxicity in Human Small Cell Lung Cancer Cells: Relationship to DNA Integrity and Topoisomerase II1

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[CANCER RESEARCH 51. 5093-5099. October I. 1991] Antagonistic Effect of Aclarubicin on Daunorubicin-induced Cytotoxicity in Human Small Cell Lung Cancer Cells: Relationship to DNA Integrity and Topoisomerase II1

Peter Buhl Jensen,2 Palle Scheide Jensen, Erland J. F. Demant, Ellen Friche, Boe Sandahl S0rensen, Maxwell Sehested, Rarsten Wassermann, Lars Vindelgv, Ole Westergaard, and Heine I IwiHansen Department of Oncology, The Finsen Institute, DK-2100 Copenhagen 0 [P. B. J., H. H. HJ; Department of Molecular Biology and Plant Physiology, University of Aarhus, DK-SOOOAarhus C fP. S. J., B. S. S., O. W.]; Department of Biochemistry C, Panum Institute, University of Copenhagen, DK-2200 Copenhagen N (E. J. F. D.I; Department of Haematology, Rigshospitalet, DK-2100 Copenhagen 0 [E. F., L. y.]; Department of Pathology, Rigshospitalet, DK-2100 Copenhagen 0 [M. S./; and Department of Biology and Toxicology, Danish National Institute of Occupational Health, Copenhagen [K. W.], Denmark

ABSTRACT increased topoisomerase II level show increased sensitivity to doxorubicin and DAU (5-7). Protein-concealed DNA breaks The effect of combinations of the anthracyclines aclarubicin and dau- are readily disclosed upon exposure to VP-16 and m-AMSA in norubicin was investigated in a clonogenic assay using the human small vivo (8, 9). In accordance with the in vivo observations, VP-16 cell lung cancer cell line OC-NYH and a multidrug-resistant (MDR) and m-AMSA stimulate topoisomerase II in vitro leading to an murine subline of Ehrlich ascites tumor (EHR2/DNR+). It was found that the cytotoxicity of daunorubicin in OC-NYH cells was antagonized elevated level of cleavable complexes (8-11). Similar findings by simultaneous exposure to nontoxic concentrations of aclarubicin. have been reported when DAU or doxorubicin has been inves Coordinate!}', aclarubicin inhibited the formation of daunorubicin-induced tigated in vitro with purified topoisomerase II and naked DNA protein-concealed DNA single-strand breaks and DNA-protein cross (1, 12-14). In whole cells, however, the formation of cleavable links in OC-NYH cells when assayed by the alkaline elution technique. protein-DNA complexes with DAU and doxorubicin is mark Aclarubicin had no influence on the accumulation of daunorubicin in these edly lower than that obtained with VP-16 at equitoxic doses cells. In contrast, the accumulation of daunorubicin in EHR2/DNR+ (15). Recently, we have reported that the anthracycline ACLA cells was enhanced by more than 300% when the cells were simultane inhibits the formation of cleavable complexes elicited by topo ously incubated with the MDR modulator verapamil, aclarubicin, or the isomerase II as well as the cleavage stimulation obtained with two agents combined. Yet the cytotoxicity of daunorubicin was potentiated VP-16 and m-AMSA (16). In whole cells, ACLA both inhibited significantly only by verapamil. The increased cytotoxicity of daunorub icin in the presence of verapamil was completely antagonized when the increase of protein-concealed DNA breaks and antagonized aclarubicin was used together with the MDR modulator. Einally, the completely the cytotoxicity of VP-16 and m-AMSA (16). Thus, effect of daunorubicin on the DNA cleavage activity of purified topoisom- ACLA may be a useful probe for investigating the cytotoxicity erase II in the presence and absence of aclarubicin was examined. It was of topoisomerase II-targeting drugs. In the present study, we found that daunorubicin stimulated DNA cleavage by topoisomerase II have examined the effect of ACLA on the cytotoxicity of DAU at specific DNA sites. The addition of aclarubicin completely inhibited in human small cell lung cancer OC-NYH cells and its effect the daunorubicin-induced stimulation of DNA cleavage. Taken together, on DNA integrity upon combined exposure of whole cells. these data indicate that aclarubicin-mediated inhibition of daunorubicin- Furthermore, we have investigated the effect of DAU on the induced cytotoxicity is due mainly to a drug interaction with the nuclear DNA cleavage activity of purified topoisomerase II in the enzyme topoisomerase II. This antagonism at the nuclear level explains why aclarubicin is a poor modulator of daunorubicin resistance even presence and absence of ACLA. Finally, we have examined the though aclarubicin is able to increase the intracellular accumulation of effect of ACLA and the resistance modulator verapamil on daunorubicin in a MDR cell line. DAU accumulation and toxicity in a murine MDR cell line. The question of ACLA combinations with DAU might be clinically relevant inasmuch as we and others have found that INTRODUCTION ACLA markedly enhances the accumulation of DAU and dox orubicin in MDR cells (17-19). There is increasing evidence that specific interactions of the "classical" anthracyclines doxorubicin and DAU1 with the nu clear enzyme topoisomerase II play a role in the cytotoxic MATERIALS AND METHODS activity of these drugs (1, 2). This is supported by the finding Drugs that resistance to the traditional topoisomerase H-targeting drugs VP-16 and m-AMSA, due either to low topoisomerase II ACLA (Lundbeck) and DAU (RhÃ´ne-Poulenc) were dissolved in levels or to altered enzyme activity, confers cross-resistance to sterile water (2 and 4 mg/ml, respectively). Verapamil (Knoll) and doxorubicin and DAU (2-4). Furthermore, cell lines with an etoposide (VP-16; Bristol-Myers) were in solution for infusion at 2.5 and 20 mg/ml, respectively. All drugs were diluted more than 100-fold Received 10/16/90; accepted 7/17/91. with RPMI 1640 supplemented with 10% fetal calf serum just prior to The costs of publication of this article were defrayed in part by the payment use. [3H]DAU (3.1 Ci/mmol) was obtained from DuPont NEN (Boston, of page charges. This article must therefore be hereby marked advertisement in MA). [m<>rA>>/-3H]Thymidine(25Ci/mmol) and [2-14C]thymidine (50 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by grants from the Danish Cancer Society, the Danish Medical mCi/mmol) were purchased from Amersham, U.K. Research Council, the Aarhus University Bioregulation Center, the Danish Na tional Agency of Technology, and the Lundbeck Foundation. Cell Lines 2To whom requests for reprints should be addressed, at the Department of Oncology, The Finsen Institute, 49 Strandboulevarden, DK-2100 Copenhagen, LI210 mouse leukemia cells were grown in suspension culture in Denmark. RPMI 1640 supplemented with 10% fetal calf serum plus penicillin 3The abbreviations used are: DAU, daunorubicin; ACLA, aclarubicin (aclaci- and streptomycin. The human small cell lung cancer cell line used was nomycin A); DPC, DNA-protein cross-link: m-AMSA, 4'-(9-acridinylamino)- OC-NYH. Source, relation to therapy, maintenance, and monitoring methanesulfon-m-anisidide; MDR. multidrug resistance: PBS. phosphate-buff ered saline (150 RIM NaCl-50 HIM phosphate. pH 7.2); SDS. sodium dodecyl have previously been described (20). The DAU-resistant subline of sulfate; SSB, DNA single-strand break; VP-16, 4'-demelhylepipodophyllotoxin- Ehrlich murine ascites tumor EHR2/DNR+ (21) was taken directly 9-(4,6-O-ethylidene-f(-D-glucopyranoside)(etoposide, VP-16-213). from mice. 5093

Clonogenic Assay DNA Cleavage Mediated by Purified Topoisomerase II

Drug toxicity was assessed by colony formation in soft agar with a Calf thymus topoisomerase II was purified according to a previously feeder layer containing sheep RBC as previously described (22). Murine described procedure (28). All experiments utilized the same DNA ascites tumor was pelleted and washed once in PBS. The cell viability fragment spanning the region from 29 to 4361 of pBR322 (1). Briefly, was assessed with dye exclusion in a hemocytometer. Single cell sus the plasmid pBR322 was digested with EcoRl. Both protruding 5' ends pensions (2 x 10" cells/ml) in RPMI 1640 supplemented with 10% were labeled with T4 polynucleotide kinase (Boehringer-Mannheim) fetal calf serum were exposed to the drugs for the indicated time at and ["P]ATP. Subsequently, a uniquely end-labeled fragment was ob 37'C and then washed twice with medium at 37Â°Cwith 5 min between tained by digestion with ///Â»ill11.The DNA fragment was incubated at washes. Cells (2 x 10') were plated to obtain 2000-3000 colonies in 30Â°Cwith 100 units of topoisomerase II in a 20-fil reaction volume the control dishes. In each experiment, the drug combinations were containing 10 mM Tris-HCl (pH 7.5), 5 mM CaCl2, 5 mM MgCl2, 0.2 tested on the same batch of cells to reduce the interexperimental HIMdithiothreitol, and 2.5% (v/v) glycerol in the presence and absence variation (20). The colonies were counted after 3 weeks of incubation. of DAU and ACLA. Following 5 min of incubation, the reactions were Accumulation of [3H|Daunorubicin terminated by the addition of SDS to 1%. The samples were digested with proteinase K. and 1 volume of deionized formamide, 0.05% bromphenol blue, 0.03% xylene cyanol, and 5 mM EDTA was added. Single cell suspensions of EHR2/DNR+ or OC-NYH cells (2 ml, 1 Subsequently, the samples were heated to 90Â°Cfor5 min before loading x 10* cells/ml) were incubated with varying concentrations of [3H] DAU for 60 min at 37"C in the presence or absence of ACLA and onto a denaturing polyacrylamide gel (6%). Hereafter autoradiography verapamil. At the end of the incubation, 10 ml of ice-cold PBS were was performed. added. Cells were then spun down at 150 x g for 5 min and washed twice with 10 ml of PBS at 4'C. To estimate drug efflux, cells were washed twice in medium, with 5-min incubations at 37Â°Cbetween RESULTS washes. Cell pellets were solubilized with 1 ml of 0.3 N HCl-50% Aclarubicin Diminishes Cytotoxicity of Daunorubicin. The ethanol and analyzed for 'H in a Packard liquid scintillation counter cytotoxic potency of DAU in the absence or presence of ACLA (17). was evaluated by clonogenic assay as described in "Materials and Methods." The results are shown in Figs. 1 and 2. Although Alkaline Elution 0.25 pM ACLA alone reduces survival to 65% of that of un DNA damage in whole cells was quantitated by alkaline elution filter treated controls, a combined treatment of OC-NYH cells with methods, as described in detail by Kohn et al. (23). Briefly, experimental cells were labeled with [2-14C]thymidine (0.02 â€ž¿Ci/ml)for72 h. L1210 ACLA and DAU for 1 h is less toxic than DAU treatment internal standard cells were labeled with [mÂ«A>>/-3H]thymidine(0.05 alone for 1 h at concentrations above 0.2 Â¿IM(Fig.\A). Thus, ACLA exerts an antagonistic effect on the cytotoxicity of DAU. fiCi/ml) for 20 h. Irradiation of cells was performed with a Therathron 780-C equipped with a ""Co source. DNA Single-Strand Breaks. [14C]Thymidine-labeled control and 100.0 drug-treated cells (5 x 10*) were mixed with an equal number of [3H] thymidine-labeled internal standard 1.1210 cells irradiated on ice with 300 rads. Hereafter the cells were suspended in PBS (10 ml at 0Â°C) and loaded onto polycarbonate filters (2-nm pore size; Nucleopore 10.0 Corp., Pleasanton, CA). Cells were washed with PBS (10 ml at 0Â°C) and lysed with 5 ml of SDS-EDTA lysis solution (2% sodium dodecyl sulfate-0.1 M glycine-0.025 M disodium EDTA) at pH 10. This was I followed by addition of 2 ml SDS-EDTA lysis solution containing 0.5 mg/ml of protdnasc K (Sigma Chemical Co., St. Louis, MO). DNA was eluted from the filters with 35 ml of tetrapropylammonium hy- droxide-EDTA, pH 12.1, containing 0.1% SDS at a rate of 0.035 ml/ min. Fractions were collected at 3-h intervals for 15 h. The fractions and filters were then processed as described in (23). DNA single-strand 0.0 0.1 0.2 0.3 0.4 break frequency expressed in rad-equivalents was calculated on the Â¿jMDAUNORUBICIN basis of first-order elution kinetics (23). DNA-Protein Cross-Links. Numbers of DNA-protein cross-links 100.00 were estimated by alkaline elution without proteinase K treatment (23, 24). Control and drug-treated [14C]thymidine-labeled OC-NYH cells were placed in iced tubes and irradiated with 3000 rads. To avoid 10.00 overloading of the DM-800 filters we loaded only 1 x IO5cells/filter as suggested (25, 26). Aliquots of 1 x 10' cells were resuspended in PBS (10 ml at 0Â°C)and loaded onto O-S-^m pore size Metrice! DM- 1.00 800 filters (Gelman Sciences, Inc., Ann Arbor, MI). Cells were washed with 10 ml of PBS and lysed with 5 ml of 2 M NaCl, 0.2% sodium

lauryl sarcosine, and 0.04 M disodium EDTA at pH 10. Residual lysis 0.100.01\B:-t."-~--V-X--.X,0.0 solution was removed by washing the filter with 0.04 M EDTA, pH 10, and the DNA was eluted from the filters overnight with tetrapropylammonium-EDTA, pH 12.1, without SDS, at a flow rate of 0.035 ml/ min. Fractions were collected at 3-h intervals for 15 h. DNA-protein 0.1 0.4Â¿jM 0.2 0.3 cross-link frequency expressed as rad-equivalents was quantitated using the bound-to-one-terminus model (27). As a control the effect of ACLA DAUNORUBICIN on VP-16-induced DNA-protein cross-links was tested. In accordance Fig. 1. .I. DAU dose-response curves obtained in a clonogenic assay on OC- NYH cells incubated for 1 h in the absence ( ) and presence ( ) of 0.25 with our previous experiments using nitrocellulose paper filters (16), MMACLA. B, DAU dose-response curves obtained on OC-NYH cells incubated ACLA was able to completely prevent VP-16-induced DNA-protein for 3 h in the absence ( ) and presence ( ) of 0.25 IIM ACLA or 0.4 JIM retention in DM-800 filters (data not shown). ACLA ( ). Points, mean of triplicate cultures: bars, SE. 5094

100.0 presence of verapamil (5 MM)is shown in Fig. 4A. ACLA at 1 MMalmost completely inhibited the cytotoxicity of 1 MMDAU. When the cells were exposed to higher ACLA concentrations ..--I the toxicity of ACLA itself became apparent. With a fixed DAU 10.0 concentration (3 UM)and varying ACLA and verapamil concen trations, similar results were obtained (Fig. 4B). In Table 1, the effects of verapamil and ACLA on [3H]DAU accumulation and efflux in EHR2/DNR+ cells are depicted. 1.0 Both verapamil (5 MM)and ACLA (1 MM)and the two drugs combined increased the accumulation of DAU to approximately 300%. While verapamil increased the accumulation of DAU by 3-fold, the efflux of DAU in the verapamil-treated cells was 0.1 also elevated when compared to cells exposed to DAU alone. o.o 0.' 0.2 0.3 0.4 0.5 0.6 0.' pM ACLARUBICIN In contrast, the amount of DAU lost in the cells exposed to ACLA was much more limited. Thus ACLA appeared to be a Fig. 2. ACLA dose-response curves obtained in a clonogenic assay on OC- more competent modulator of DAU efflux than verapamil. NYH cells incubated for 3 h. Effect of ACLA in the absence ( ) and presence Aclarubicin Reduces Daunorubicin-induced DNA Single- ( ) of 0.06 JIMDAU. Poinls, mean of triplicate cultures; bars, SE. Strand Breaks and DNA-Protein Cross-Links. To ascertain whether drug-mediated cytotoxicity was accompanied by pro Fig. \B demonstrates the result of prolonging the exposure duction of DNA SSBs within OC-NYH cells, the DNA single- time to 3 h. This prolonged drug exposure resulted in an strand cleaving activity of the drugs was assessed using the approximate 5-fold higher cytotoxicity of DAU. Also, the an alkaline elution filter method. Experiments were performed tagonistic effect of ACLA was amplified at prolonged drug after treatment of whole cells with either DAU or ACLA or a exposure. Thus, the combination treatment with ACLA and combination of these two drugs for 3 h at 37Â°C.Both drugs DAU resulted in a marked upward shift of the dose-response produced DNA SSBs (Fig. 5). A 2.5-fold enhancement of the curve, with a 10-100-fold increase in survival. The effect of SSB frequency was observed, however, in the DAU-treated cells increasing concentrations of ACLA on the cytotoxic activity of compared with the ACLA-treated cells (Fig. 5 and Table 2). a fixed DAU concentration is shown in Fig. 2. ACLA concen trations above 0.25 IÂ¿Msignificantly inhibit the cytotoxic effect 100 of DAU, and at 0.6 UM ACLA the cytotoxicity of DAU is almost entirely antagonized. As the toxicity of ACLA alone is enhanced with longer drug exposure times (29), the duration of drug exposure was not increased further. Aclarubicin Increases the Accumulation of Daunorubicin in MDR Cells but Antagonizes Its Cytotoxicity. In accordance with our previous studies on wild-type cell lines (17), ACLA (0.25 UM) displayed no influence on the accumulation of [3H]DAU in OC-NYH cells (data not shown). In contrast, we and others have previously demonstrated that aclarubicin increases the accumulation of DAU and doxorubicin in MDR cells, an effect similar to that of verapamil (17-19). We therefore examined 1 1 r the influence of ACLA and verapamil on DAU cytotoxicity and (100 3 2 10/uM4 6 8 accumulation in the MDR cell line EHR/DNR+. Fig. 3A shows DAUNORUBICIN\ DAU cytotoxicity in the cell line EHR2/DNR+, which is primarily resistant to DAU. In these cells DAU is almost 001pE nontoxic up to 10 UMafter 1 h of treatment. Exposure to 1 /Ã•M 4...\v, â€¢¿-' ACLA is nontoxic in this cell line, and only a very limited R cytotoxicity was observed when ACLA was combined with C10.00E DAU. In contrast, a clear modulation of resistance to DAU is NTS ',>Vk1 obtained when cells were simultaneously exposed to a nontoxic 1.00uRvA dose of 5 UM verapamil. This increase in DAU toxicity was antagonized when ACLA was combined with verapamil. The dose-response curve obtained with verapamil shows saturation 1\^ Â°JÂ°L1 (becomes horizontal) at high DAU concentrations, and the maximum effect of verapamil on DAU cytotoxicity was a reduction of survival to 20-30% when compared to the control. 0.01. 1 In order to achieve a higher cytotoxic effect of DAU, the 01 2345 incubation time was prolonged. Fig. 3B shows the effect of a 3- pM DAUNORUBICIN h DAU incubation. As observed with OC-NYH cells (Fig. 1, A Fig. 3. l. DAU dose-response curves obtained in a clonogenic assay on EHR2/ and B), ACLA antagonized the effect of DAU more efficiently DNR+ cells incubated for 1 h. , DAU alone; , DAU + 1.0 Â»iMACLA; under these conditions. Thus, at 0.5 MMDAU the difference in â€”¿,DAU 4- 5 /Â¡Mverapamil; , DAU + 1.0 >iM ACLA and 5 MM toxicity is more than 20-fold. verapamil. B, DAU dose-response curves on EHR2/DNR+ cells incubated for 3 h. , DAU alone; , DAU + 1.0 /iM ACLA; , DAU + 5 fiM The effect of increasing concentrations of ACLA on the verapamil; , DAU + 1.0 MMACLA and 5 JIM verapamil. Poinls, mean of cytotoxic activity of three different DAU concentrations in the triplicate cultures; bars, SE. 5095

100.0 resulted in a significant inhibition of drug-mediated SSBs (Ta ble 2 and Fig. 5). As such, addition of 0.3 UM ACLA to 1 ^M DAU resulted in a 30% reduction of SSB frequency when compared to the break frequency in cells treated with 1 /JM DAU alone. A comparable effect of ACLA on DAU-mediated SSBs was also observed in LI210 cells (data not shown). Finally, the production of DAU-mediated DPCs was signifi cantly inhibited by ACLA in a dose-dependent manner (Table 2). Thus, addition of 0.3,0.5, and 1.0 Â¿IMACLAto 1 ^M DAU, respectively, reduced the DAU-mediated DPCs to 55%, 49%, and 45% of what was observed with DAU alone. Aclarubicin Inhibits Daunorubicin-induced Topoisomerase II 0.1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Cleavage. The effect of DAU on purified calf thymus topoisomerase II-mediated DNA cleavage of a 32P-labeled DNA fragment ÃŸMACLARUBICIN was studied in the absence and presence of ACLA. Addition of 100.0 DAU in the concentration range from 10 nM to 1 n\\ induced P new topoisomerase II-mediated DNA cleavage sites and stim- E R C N 10.0

Control 1.0 0.3 MM ACLA

3.4 MM DAU 0.11 B 01234567 20 MM ACLA >0.7 pM DAU pM ACLARUBICIN 0.7pM DAU Â¥ Fig. 4. A, ACLA dose-response curves obtained in a clonogenic assay on 0.3MM ACLA. 1.0MM DAU EHR2/DNR+ cells incubated for 2 h in the presence of 5 Â¿IMverapamiland in 1.0 DAU the absence ( ) and presence of 1.0 UM( ). 3.0 JIM( ), and 5.0 /JM 10- ( ) DAU. B, ACLA dose-response curves on EHR2/DNR+ cells incubated 100 90 80 70 60 50 40 30 20 for 2 h in the presence of 3 UMDAU and in the absence ( ) and presence of 5 L1210 RETENTION (%) Â¡Ã•Ã•A( ), 10 MM ( ). and 15 JIM verapamil ( ). Points, mean of triplicate cultures; ears, SE. Fig. 5. Representative alkaline elution curves for DNA single-strand break production induced by DAU in the presence and absence of 0.3 /JMACLA in OC- NYH cells. Treatment was for 3 h at 37'C with quantification of DNA cleavage immediately following the drug exposure (see "Materials and Methods"). Table 1 Accumulation and efflux off'HJdaunorubicin in the multidrug-resistant EHR2/DNR+ cell line incubated with or without verapamil and aclarubicin pmol |3H]daunorubicin/10' cells Table 2 DNA SSB formation and DPCs in OC-NYH cells incubated for 3 h with DAU + DAU + DAU + VER the indicated drugs DAU" VER ACLA + ACLA DNA single-strand DAU accumulation 146(100%)' 456(312%) 446(305%) 472(323%) Drug i

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1991 American Association for Cancer Research. ACLARUBICIN INHIBITION OF DAUNORUB1CIN CYTOTOXICITY ulated preexisting cleavage sites when compared to the topoi- ABCDEFGHI JKLMN somerase II mediated DNA cleavage in the absence of drug (Fig. 6, Lanes C-E). The maximum stimulatory effect of DAU on topoisomerase H-mediated DNA cleavage was observed at 100 nM (Fig. 6, Lane D). The enzyme was then incubated with its DNA substrate for 5 min in the presence of ACLA (1 ^M) WH111 before addition of DAU. The preincubation with ACLA inhib ited the stimulation seen with DAU (Fig. 6, Lanes F-H). Furthermore, ACLA administered by itself inhibited almost all topoisomerase II mediated DNA cleavages when present in concentrations >1 Â¿tM.However, close examination of Fig. 6 reveals a stimulatory effect with the lower concentrations of ACLA (1-10 nM; Fig. 6, Lanes I and /). However, the weak stimulatory effect of ACLA is in contrast to the pronounced stimulatory effect observed with DAU.

DISCUSSION

The present study demonstrates that ACLA is able to inhibit the cytotoxicity of DAU both in a sensitive cell line and in a MDR cell line made sensitive to DAU by the use of the resistance modulator verapamil. In accordance with our pre vious findings of the effect of ACLA on the cytotoxicity of VP- 16 (16), ACLA exhibited an antagonistic effect on DAU cyto toxicity in OC-NYH cells after a 1-h drug exposure. However, in contrast to VP-16 (31) a substantial fraction of DAU is retained in cells after repeated washes with PBS or RPMI 1640 supplemented with 10% fetal calf serum. Thereby a significant amount of DAU is plated in the clonogenic assay (Table 1 and Ref. 15). Accordingly, the enhanced antagonistic effect observed with a 3-h incubation (Fig. IB) could be due to a decrease in the influence of DAU retention as a larger fraction of cells are killed within the intended exposure time. In contrast to the observations with the OC-NYH cells, the DAU dose-response Fig. 6. Effect of DAU on topoisomerase II-mediated DNA cleavage. Reaction mixtures containing a "P-labeled DNA fragment and purified topoisomerase II curve in EHR2/DNR+ cells became horizontal after an initial were incubated with various drugs before addition of SDS. Samples were treated exponential cell kill (Fig. 3). An efficient cellular DAU efflux with proteinase K and then subjected to electrophoresis in a polyacrylamide gel. should result in a saturable dose-response curve since the effect Lane A, control without enzyme; in all other lanes topoisomerase II was added. of DAU is cell-cycle specific (20, 32). Thus, the saturable DAU I-une B, baseline topoisomerase II formation of DNA cleavage; Lanes C-E, result of incubation with increasing concentrations of DAU; Lane C, 10 nM; Lane D, dose-response curves indicate that drug retention after plating 100 nM; Lane E, I ^M DAU. Lanes F-H, result of incubating ACLA (1 ,iM) was without influence on the cytotoxicity obtained in EHR2/ together with DAU. Lane F, 10 nM; Lane G, 100 nM; Lane H, l Â»MDAU. Lanes I-N, effect of ACLA on the cleavage reaction. Lane I, 1 nM; Lane], 10 n\i; Lane DNR+ cells. K, 100 nM; Lane L, 1 ^M; Lane M, 10 >IM;Lane N, 100 ^M ACLA. In OC-NYH cells we found that DAU-induced DNA single- strand breaks (Table 2) increased up to 1.3 MMDAU and then reached their maximum level at 400-450 rad equivalents at whereas enzyme-mediated DNA cleavage was suppressed at 1.3-1.9 MM.When incubating OC-NYH cells with 1.0 MMDAU higher drug concentrations. Thus it appears that DNA inter for 3 h we found a SSB:DPC ratio of 1:1 (Table 2), suggesting calating agents can inhibit topoisomerase II activity by two that all DAU-induced single-strand breaks were protein con different mechanisms: (a) by poisoning the enzyme by trapping cealed. These results are in agreement with those of Belvedere it on the DNA as a cleavable complex and (!>)by inhibiting the et al. (33), who found that all daunorubicin-mediated single- enzyme from binding to DNA. The DAU stimulation of cleav strand breaks were protein concealed in LoVo cells. Previously able complex formation in vitro could be completely inhibited we demonstrated that ACLA was able to inhibit the formation by ACLA coincubation. This observation agrees with our find of protein-concealed DNA breaks obtained with VP-16 and m- ings concerning the effect of ACLA on VP-16- and m-AMSA- AMSA in OC-NYH cells. In accordance with this, the present stimulated topoisomerase II-mediated DNA cleavages and with paper demonstrates that ACLA was able to decrease the for the bell-shaped stimulation/inhibition curve observed with mation of DAU-induced protein-concealed DNA breaks in a DAU. dose-dependent manner (Table 2). It is possible that the antagonistic effect of ACLA on DAU DNA intercalation is clearly not sufficient for generation of cytotoxicity, instead of being due to a direct interaction with topoisomerase II-DNA complexes (1). Although both DAU the topoisomerase II substrate, is caused by inhibition of RNA and ACLA intercalate into DNA (34), only DAU exhibited a synthesis exhibited by ACLA (35). ACLA is a potent inhibitor profound stimulation of topoisomerase II-mediated cleavage of RNA synthesis (36). A direct effect of ACLA on the topoi (Fig. 6). In agreement with others (12, 13) the effect of DAU somerase II substrate is suggested, however, when examining on topoisomerase II-mediated DNA cleavage was "bell shaped," the results obtained on OC-NYH with, e.g., 0.3 MMACLA. As since lower concentrations of DAU stimulated DNA cleavage, seen in Figs. IB and 2 this concentration clearly antagonized 5097

-resistant P388 leukemia cell lines. Cancer Res., 49: 58-62, 1989. the cytotoxicity of DAD. Coordinately, the same dose of ACLA Glisson, B., Gupta. R.. Hodges, P., and Ross, W. Cross-resistance to inter diminished the formation of SSBs and DPCs produced by DAU calating agents in an epipodophyllotoxin-resistant Chinese hamster ovary (Fig. 5 and Table 2). Thus, from these data it seems most likely cell line: evidence for a common intracellular target. Cancer Res.. 46: 1939- that the effect of ACLA was exerted at the level of topoisom- 1942. 1986. Danks, M. K., Schmidt. C. A., Cirtain, M. C., Suttle. D. P., and Beck, W. erase II. This is also indicated by the inhibitory effect of ACLA T. Altered catalytic activity of and DNA cleavage by DNA topoisomerase II on DNA cleavage mediated by purified topoisomerase II. from human leukemic cells selected for resistance to VM-26. Biochemistry. 27:8861-8869. 1988. Although circumstantial, the present results point to topoi Robson, C. N., Hoban, P. R., Harris, A. L., and Hickson, I. D. Cross- somerase II as a major target of DAU. On the other hand the sensitivity to topoisomerase II inhibitors in cytotoxic drug-hypersensitive effect of ACLA is not confined to topoisomerase II. Thus the Chinese hamster ovary cell lines. Cancer Res., 47: 1560-1565, 1987. Davies, S. M., Robson. C. N., Davies, S. L., and Hickson, I. D. Nuclear inhibition of topoisomerase II by ACLA is similar to the effects topoisomerase II levels correlate with the sensitivity of mammalian cells to of the minor groove binders distamycin and Hoechst 33258 intercalating agents and epipodophyllotoxins. J. Biol. Chem.. 263: 17724- (37). The minor groove binders also inhibit topoisomerase I 17729, 1988. Tan, K. B., Mattern, M. R., Boyce. R. A., and Schein. P. S. Elevated DNA (38, 39), and ACLA can possibly be regarded as a minor groove topoisomerase II activity in nitrogen mustard-resistant human cells. Proc. binder with the three sugar groups placed in the minor groove Nati. Acad. Sci. USA, 84: 7668-7671, 1987. Yang, L., Rowe, T. C., and Liu, L. F. Identification of DNA topoisomerase (34, 40). II as intracellular target of antitumor epipodophyllotoxins in simian virus Cross-resistance to epipodophyllotoxins, anthracyclines, and 40-infected monkey cells. Cancer Res., 45: 5872-5876, 1985. Vinca alkaloids is a hallmark of the MDR phenotype in cells Rowe, T. C., Chen, G. L., Hsiang, Y-H., and Liu, L. F. DNA damage by antitumor acridines mediated by mammalian DNA topoisomerase 11.Cancer expressing P-glycoprotein, whereas resistance to Vinca alka Res., 46:2021-2026, 1986. Nelson, E. M., Tewey, K. M.. and Liu, L. F. Mechanism of antitumor drug loids is not included in the altered topoisomerase MDR phe action: poisoning of mammalian DNA topoisomerase II on DNA by 4'-(9- notype (41). In agreement with this observation, ACLA has no acridinylamino)-melhanesulfon-m-anisidide. Proc. Nati. Acad. Sci. USA, */: inhibitory effect on the cell kill obtained with vincristine in OC- 1361-1365, 1984. NYH cells.4 The inhibitory effect of ACLA on DAU cytotox Bodley, A. L., and Liu, L. F. Roles of DNA topoisomerases in drug cytotox icity and drug resistance. In: P. V. Wooley and K. D. Tew (eds.), Mechanisms icity explains the nonexistent or limited synergy that is obtained of Drug Resistance in Neoplastic Cells, pp. 277-286. San Diego, CA: when ACLA increases DAU uptake in MDR cells. Academic Press, Inc., 1988. A question arising from the present study is whether the 12. Capranico, G., Zunino, F., Kohn, K. W., and Pommier, Y. Sequence-selective topoisomerase II inhibition by anlhracycline derivatives in SV40 DNA: effect of ACLA on DAU transport in MDR cells and the relationship with DNA binding affinity and cytoloxicity. Biochemistry, 29: inhibition of DAU action on its target can be separated. ACLA 562-569, 1990. 13. Bodley, A.. Liu. L. F., Israel, M., Seshadri. R.. Koseki. Y., Giuliani, F. C, is more lipophilic than DAU and accumulates rapidly to the Kirschenbaum. R.. Silber, R., and Potmesil, M. DNA topoisomerase II- same levels in wild-type and MDR Ehrlich cells (42, 43). mediated interaction of doxorubicin and daunorubicin congeners with DNA. However, the lack of net ACLA efflux in the MDR cells does Cancer Res., 49: 5969-5978, 1989. 14. Wassermann, K., Markovits, J., Jaxel, C., Capranico, G., Kohn, K. W., and not necessarily mean that ACLA is not a substrate for P- Pommier, Y. Effects of morpholinyl doxorubicins, doxorubicin, and actino- glycoprotein. The large increase in DAU accumulation obtained mycin D on mammalian DNA topoisomerases I and II. Mol. Pharmacol., with ACLA indicates inhibition of DAU efflux in EHR2/ JÂ«:38-45, 1990. 15. Binaschi, M., Capranico, G., De Isabella, P., Mariani, M., Supino, R., Tinelli, DNR+ (Ref. 17 and Table 1), and ACLA is even more effective S., and Zunino, F. 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In vitro circumvention of anthracycline-resistance in Ehrlich ascites tumour by an- cleavage displaying the same effect as ACLA but showing a net thracycline analogues. Biochem. Pharmacol., 39: 1721-1726, 1990. transport out of MDR cells could be used in combination with 18. Millot, J-M.. Rasoanaivo. T. D. W., Morjani. H., and Manfait. M. Role of a typical topoisomerase II-targeting drug for selective kill of the aclacinomycin A-doxorubicin association in reversal of doxorubicin re sistance in K562 tumor cells. Br. J. Cancer, 60: 678-684, 1989. MDR cells. Thus, in a population of MDR tumor cells, e.g., 19. Tapiero, H.. Boule. D., Trincai, G., Fourcade, A., and Lampidis, T. J. relapsed leukemia cells, the MDR-transported ACLA analogue Potentiation of Adriamycin accumulation and effectiveness in Adriamycin- would be effluxed from the tumor cells while the "sensitive" resistant cells by aclacinomycin A. Leuk. Res., 12:411-418. 1988. 20. Jensen, P. B., Roed, H.. Vindelev. L.. 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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1991 American Association for Cancer Research. Antagonistic Effect of Aclarubicin on Daunorubicin-induced Cytotoxicity in Human Small Cell Lung Cancer Cells: Relationship to DNA Integrity and Topoisomerase II

Peter Buhl Jensen, Palle Schelde Jensen, Erland J. F. Demant, et al.

Cancer Res 1991;51:5093-5099.

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