Antagonistic Effect of Aclarubicin on the Cytotoxicity

[CANCER RESEARCH 50. 3311-3316. June 1. 1990) Antagonistic Effect of Aclarubicin on the Cytotoxicity of Etoposide and 4'- (9-Acridinylamino)methanesulfon-w-anisidide in Human Small Cell Lung Cancer Cell Lines and on Topoisomerase II-mediated DNA Cleavage1

Peter Buhl Jensen,2 Boe Sandahl S0rensen, Erland J. F. Demant, Maxwell Sehested, Palle Scheide Jensen, Lars Vindel0v, and Heine I loi Hansen Department of Oncology, The Finsen Institute, DK-2100 Copenhagen Q[P. B. J., L. K, H. H. H.]; Department of Molecular Biology and Plant Physiology, University ofAarhus, DK-8000 Aarhus C [B. S. S., P. S. J.]; Department of Biochemistry C, university of Copenhagen. Panum Institute, DK-2200 Copenhagen N Â¡E.J. F. D./; and Department of Pathology, Rigshospitalet, DK-2100 Copenhagen

ABSTRACT cleavable complexes plays a role in the toxicity of these drugs too (6, 7). Aclarubicin intercalates into DNA like Adriamycin The effect of combinations of the anthracycline aclarubicin and the topoisomerase II targeting drugs 4'-demethylepipodophyllotoxin-9-(4,6- and daunorubicin (8); however, to our knowledge, there are no O-ethylidene-/3-D-glucopyranoside) (VP-16) and 4'-(9-acridinylam- reports on aclarubicin stimulation of the formation of cleavable ino)methanesulfon-m-anisidide (m-AMSA) was investigated in a clono- complexes. The different sensitivity patterns to Adriamycin and genic assay. The cytotoxicity of VP-16 was almost completely antago aclarubicin observed in panels of cell lines by us and others (9- nized by preincubating cells with nontoxic concentrations of aclarubicin. 12) indicate that the two drugs may differ in their mechanism The inhibition of cytotoxicity was not seen when the cells were exposed of action. Furthermore we have found that aclarubicin, com to aclarubicin after exposure to VP-16. The inhibition was significant pared with Adriamycin, becomes relatively more potent with a over a wide range of aclarubicin concentrations (3 u\i to 0.4 MM),above prolonged drug exposure time (12) and that the cytotoxicity of which the toxicity of aclarubicin became apparent. A similar effect was aclarubicin, in contrast to Adriamycin, daunorubicin, and mi- seen on the toxicity of m-AMSA. In contrast to aclarubicin, preincubation toxantrone, is independent of the proportion of cells in S-phase with Adriamycin did not antagonize the effect of VP-16. With purified at the time of drug exposure (13). Intercalation into DNA topoisomerase II and naked DNA, aclarubicin did not stimulate the formation of cleavable complexes between topoisomerase II and DNA. without stimulation of the formation of cleavable complexes is Aclarubicin concentrations above 1 MMinhibited the baseline formation seen with the cytotoxic compound ethidium bromide (14, 15). of cleavable complexes elicited with the enzyme alone. Low (1 to 10 n\i) Furthermore ethidium bromide inhibits the baseline formation aclarubicin concentrations increased the formation of cleavable com of cleavable complexes elicited by topoisomerase II alone as plexes obtained with VP-16 and m-AMSA; however, at aclarubicin well as the stimulation seen with the classical topoisomerase II concentrations above 1 MMan antagonistic effect was obtained. In cells, targeting drugs VP-16 and m-AMSA. In accordance with these the m-AMSA- and VP-16-induced, protein-concealed DNA strand breaks findings ethidium bromide is able to inhibit the cytotoxicity of were completely inhibitable by aclarubicin preincubation with no synergic VP-16 in a clonogenic assay (15). In this study we used a dose levels. Our results suggest that aclarubicin inhibits topoisomerase clonogenic assay to investigate if aclarubicin was able to inhibit II-mediated DNA cleavage. This inhibition could represent the mecha nism of action of the drug and explain the lack of cross-resistance to the the toxicity of topoisomerase II targeting drugs. We further classical anthracyclines. The observed antagonism could have conse investigated the effect of aclarubicin on the formation of cleav quences for scheduling of aclarubicin with topoisomerase II-active anti- able DNA-protein complexes in cells as well as with purified topoisomerase II and a 12P-labeled DNA fragment. cancer drugs.

INTRODUCTION MATERIALS AND METHODS Aclarubicin as well as m-AMSA-1 and VP-16 are cytostatic Drugs. For use in the clonogenic assay, aclarubicin (Lundbeck) and drugs with documented activity in some human neoplasms as, Adriamycin (Farmitalia Carlo-Erba) were dissolved in sterile water (1 for instance, acute myelocytic leukemia (1). Clinical trials with mg/ml). Etoposide (VP-16) (Bristol-Myers) and m-AMSA (Parke- combinations of these drugs are therefore attractive (2), espe Davis) were in solution for infusion at 20 mg/ml and 50 mg/ml, cially as aclarubicin seems to be non-cross-resistant to the first respectively. All drugs were diluted more than 100-fold with tissue line anthracyclines Adriamycin and daunorubicin (3). Both VP- culture medium just prior to use. In in vitro studies with purified 16 and m-AMSA are "classical" DNA topoisomerase II target topoisomerase II, VP-16 (Bristol-Myers) and m-AMSA (Parke-Davis) were dissolved in DMSO and aclarubicin (Lundbeck) in water. [I4C]- ing drugs. Their mechanism of toxicity seems to be linked to a VP-16 (18 MCi/mg) was a generous gift from Bristol-Myers, Syracuse, stimulation in the formation of cleavable complexes between NY. [miiAy/-'H]Thymidine (25 Ci/mmol) was from Amersham, United DNA and topoisomerase II (4, 5). Although other mechanisms Kingdom. of action may be involved in the toxicity of Adriamycin and Cell Lines. The human cell lines used were OC-NYH (also designated daunorubicin, there is increasing evidence that formation of GLC-2) and OC-TOL (GLC-3). Their source, relation to therapy, maintenance, and monitoring have previously been described (13). Received 9/11/89; revised 1/30/90. The costs of publication of this article were defrayed in part by the payment Clonogenic Assay. Drug toxicity was assessed by colony formation of page charges. This article must therefore be hereby marked advertisement in in soft agar with a feeder layer containing sheep red blood cells as accordance with 18 U.S.C. Section 1734 solely to indicate this fact. previously described (16). Single cell suspensions (2 x IO4cells/ml) in 1Supported by grants from the Danish Cancer Society and from the Lundbeck RPMI 1640 supplemented with 10% fetal calf serum were exposed to Foundation. the drugs and then washed twice with PBS at 20Â°C.Cells (2 x 10") 1To whom requests for reprints should be addressed, at the Department of Oncology. The Finsen Institute. 49 Strandboulevarden, DK-2100 Copenhagen. were plated to obtain 2000 to 3000 colonies in the control dishes. In Denmark. each experiment the drug combinations were tested on the same batch 3The abbreviations used are: m-AMSA. 4'-(9-acridinylamino)methanesulfon- of cells to reduce the interexperimental variation (13). The colonies m-anisidide; DMSO. dimethyl sulfoxide; PBS, phosphate-buffered saline (150 were counted after 3 wk of incubation. mM NaCI:50 mm phosphate, pH 7.2); SDS, sodium dodecyl sulfate; SCCL, small cell carcinoma of the lung; VP-16. 4'-demethylepipodophyllotoxin-9-(4.6-O- Accumulation of |I4C1VP-16. Single cell suspensions of OC-NYH ethylidene-J-D-glucopyranoside) (etoposide. VP-16-213). cells (1 ml. 5 x 10" cells) were incubated with [14C]VP-16 (10 MM)for 3311

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. ACLARIJBICIN INHIBITION OF VP-16 AND m-AMSA TOXIC1TY 15 or 60 min at 37Â°Cin the presence or absence of varying concentra tions of aclarubicin added 20 min before [I4C]VP-16. At the end of the incubation. 10 ml of ice-cold PBS were added as this procedure has been shown to stop VP-16 fluxes (17). Cells were then spun down at 150 x g for 5 min, washed twice with 10 ml of ice-cold PBS, solubilized with 0.2 M NaOH. and analyzed for UC in a Packard liquid scintillation spectrometer (17). DNA Cleavage Mediated by Purified Topoisomerase II. The calf thymus topoisomerase II used was purified according to a previously described procedure (18). All experiments utilized the same Tetrahy- mena termophila rDNA fragment spanning the region from -967 to â€”635with respect to the initiation point of transcription. This rDNA fragment was isolated from the plasmid pUTtrll by excision with the restriction enzymes Bam\\\ and ///ndlll. Finally the isolated fragment was "P labeled at both 5' ends (Fig. 6. bottom) (18). Labeling both 5' 10 15 20 ends made it possible to examine the topoisomerase II cleavage reaction uM VP-16 on both DNA strands as topoisomerase II cleavage gives rise to a short Fig. 1. VP-16 dose-response cunes obtained in a clonogenic assay with cell labeled fragment from the noncoding strand and a longer labeled line OC-NYH. The cells were incubated for 60 min with ( ) and without fragment from the coding strand. The "P-labeled DNA fragment (20 ( ) preincubalion for 20 min with 0.4 MMaclarubicin. Points, mean: bars, SE pmol) was incubated with 100 units of topoisomerase II in a 20-^1 from triplicate cultures. reaction volume containing 10 IHMTris-HCl (pH 7.5), 5 HIMCaCl2, 5 mM MgCI;, 0.2 mM dithiothreitol. 2.5% (v/v) glycerol, and m-AMSA, 100.0 VP-16, aclarubicin, or combinations of these compounds. In all reac tions the final concentration of DMSO was 5% (v/v). Aclarubicin incubations were performed at 30Â°Cfor 5 min prior to addition of m- AMSA or VP-16. Following 5 min of incubation, the reactions were 10.0 terminated by the addition of SDS to a final concentration of 1% (v/ v). The samples were then digested with 250 /jg/ml of proteinase K at 37*C for 30 min before addition of 1 volume of deionized formamide with 0.05% bromphenol blue, 0.03% xylene cyanol, and 5 mM EDTA. Subsequently, the samples were heated to 90Â°Cfor5 min before loading on a denaturing polyacrylamide gel (6%). Autoradiography was per formed at -70Â°Cwith Fuji RX film and a Du Pont intensifying screen. "--I Determination of Protein-DNA Cross-Links. Human lymphoblastoid Daudi cells grown in RPMI 1640 with 10% fetal calf serum were labeled with ['HJthymidine (2.5 Â¿iCi/ml)for 24 h. The cells were 0.1 10 15 20 25 30 35 40 incubated for 30 min with 100 pM to 100 ^M aclarubicin, followed by lM VP-16 incubation for 30 min with either 10 n\\ m-AMSA, 1 /Â«Mm-AMSA, 60 ÃÃMVP-16,or 6 MMVP-16. Approximately 1 x IO1cells in 50 Â¿ilof Fig. 2. VP-16 dose-response curves obtained on OC-NYH cells incubated for 60 min with (---- ) and without (- ) preincubation for 20 min with 0.2 JIM medium were lysed in 500 ^1 of 1.25% SDS:2.5 mM EDTA:0.125 M Adriamycin. Points, mean: bars. SE from triplicate cultures. NaOH and added to a nitrocellulose filter (Sleicher & Schuell, Dassel, Federal Republic of Germany) preincubated with 1 ^g/ml of salmon sperm carrier DNA. The filter was washed twice with I ml of 100 mM 100 + NaOH:l mM EDTA and soaked in 96% ethanol. Protein-['H)DNA pER cross-links were determined by counting in a Beckman scintillation 1^^-H^â€¢-^//i""; counter. The total amount of ['HjDNA was determined by assessing C E the amount of trichloroacetic acid-insoluble |'H]DNA in the flow- N through from the nitrocellulose filter. TS 10URV1V /.'/ff/ RESULTS /,-â€¢-"0.0,1 .L To investigate the effect on cytotoxicity of combining acla AL1^\,____* rubicin and VP-16, SCCL cells were incubated with aclarubicin (0.4 Â¿IM)for20 min prior to a 60-min treatment with varying concentrations of VP-16. At the end of the drug treatment 0.1 0.20.7uM 0.3 0.4 0.5 0.6 period, both drugs were removed by washing the cells twice in ACLARUBICIN drug-free PBS, and the cells were then seeded in soft agar to Fig. 3. Dose-response curves obtained on OC-NYH cells incubated with assess colony-forming ability. VP-16 dose-response curves ob increasing aclarubicin concentrations for 80 min without VP-16 (- ). preincu tained on the cell line OC-NYH with or without aclarubicin bation for 20 min with aclarubicin and 60-min incubation with both aclarubicin and VP-16 (40 Â¿I.MVP-16,--- ) and 60 /JM VP-16. ---- ). Sixty-min treatment are shown in Fig. 1. Aclarubicin alone reduced the incubation with 40 >JMVP-16 and thereafter 20 min of postincubation with survival to 80% (SE = 4%). The lethal effect of VP-16 was aclarubicin ( ..... ). Points, mean: bars. SE from triplicate cultures. almost completely inhibited by aclarubicin, and at 40 UM VP- 16, aclarubicin increased cell survival from about 1% to 50%. additive cell kill was obtained with all tested VP-16 concentra Similar results were obtained with the cell line OC-TOL (data tions. not shown). The experiments were repeated with Adriamycin In subsequent experiments on the cell line OC-NYH, the preincubation at a concentration (0.2 n\\) equitoxic to 0.4 Â¿IM aclarubicin concentration was varied at a fixed (40 or 60 Â¿IM) aclarubicin [survival reduced to 79% (SE = 2%)]. As seen from concentration of VP-16. As can be seen from Fig. 3, all acla Fig. 2, Adriamycin could not inhibit the VP-16 toxicity, and an rubicin concentrations tested (0.1 to 0.6 Â¿Â¡M)reducedthe tox- 3312

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. ACLARUBICIN INHIBITION OF VP-I6 AND m-AMSA TOX1CITY icity of VP-16. Nearly full protection of the cells toward VP- 100.0 16 was reached at 0.4 ^M aclarubicin. A much lower degree of protection was obtained when the cells were exposed to VP-16 before the incubation with aclarubicin (data included in Fig. 3). To investigate if the prevention of VP-16 toxicity by aclarubicin 10.0 could be a result of an aclarubicin influence on the cellular accumulation of VP-16, the accumulation of [I4C]VP-16 in OC- NYH cells was measured with and without preincubation of the cells with aclarubicin. The results are shown in Table 1. The VP-16 accumulation at 15 and 60 min was unaffected by 0.1, 0.4, and 0.8 ÃŸMaclarubicin preincubation. Thus, aclarubicin had no significant effect on the accumulation of VP-16.

Preincubation of OC-NYH cells with aclarubicin (0.4 /UM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 was found also to protect against the intercalating drug m- pM ACLARUBICIN AMSA (Fig. 4). In Fig. 5, dose-response curves to aclarubicin Fig. 5. Dose-response curves obtained on OC-NYH cells incubated with alone and aclarubicin plus m-AMSA (5, 10, and 15 ÃŸM)are increasing aclarubicin concentrations for 80 min without m-AMSA ( ), prein cubation for 20 min with aclarubicin and 60-min incubation with both aclarubicin shown. Protection was obtained at all tested doses, although and m-AMSA (5 MMm-AMSA, ; 10 MMm-AMSA. ; and 15 MMm- the aclarubicin toxicity becomes visible at concentrations above AMSA, ). Points, mean: bars, SE from triplicate cultures. 0.3 ÃŸM,resulting in biphasic dose-response curves. The effect of aclarubicin on topoisomerase II-mediated DNA A BCDEFG HIJKLMN OPQRSTU cleavage was studied in vitro by reacting highly purified calf thymus topoisomerase II with a 32P-labeled DNA fragment. Incubating this fragment with purified topoisomerase II dem onstrated that the enzyme has a very specific interaction with its DNA substrate, as only a single major cleavage was intro duced (Fig. 6, Lanes B, H, and O). Consistent with previously presented data (18), the enzyme had a marked preference for cleaving the noncoding (nc) as compared with the coding (c) strand. Addition of aclarubicin in concentrations of l ÃŸMor higher abolished topoisomerase II cleavage (Fig. 6, Lanes Fand G). However, at concentrations below 1 ^M, the DNA cleavage performed by topoisomerase II was unaffected by aclarubicin (Fig. 6, Lanes C, D, and E). This demonstrated that aclarubicin

Table 1 Effect of aclarubicin on cellular accumulation of VP-16 â€¢ Accumulation of |'"C]VP-16 in OC-NYH cells was measured after 15- and 60- min incubations with 10 MMVP-16. Prior to VP-16 addition, the cells were incubated for 20 min with or without aclarubicin. I -- VP-16 accumulation (pmol/106 cells)

min18.0 min22.1 TopoÃ¯Ã¯ VP-16 Â±0.9Â° Â±1.4 + 0.1 MMaclarubicin 17.9 Â±0.7 23.5 Â±0.6 + 0.4 MMaclarubicin 19.2 Â±0.2 25.9 Â±0.4 Fig. 6. Effect of aclarubicin on topoisomerase II-mediated DNA cleavage. Reaction mixtures containing a 32P-labeled DNA fragment and purified topoisom + 0.8 MMaclarubicin15 19.2 Â±0.460 24.6 Â±1.2 1Mean Â±SD of three determinations. erase II were incubated with various drugs before addition of SDS. Samples were incubated with proteinase K and then subjected to electrophoresis in a polyacrylamide gel. Lane A is a control without enzyme. Lanes B, H, and O show the baseline topoisomerase II ( Topo li) formation of DNA cleavage in the absence of 100 drug. Lanes C to G are the result of incubation with increasing concentrations of aclarubicin; Lane C, 1 nM; Lane D, 10 nM; Lane E, 0.1 MM;Lane F, 1 MM;and Lane G, 10 MMaclarubicin. In Lanes 1 to N, 10 MMm-AMSA was added (Lane 1, without aclarubicin preincubation; Lanes J to N, with increasing concentrations of aclarubicin). Lane J, 10 nw; Lane K, 0.1 MM;Lane L, 1.0 MM;Lane M, 10 MM; Lane N, 100 MMaclarubicin. In Lanes P to U, 60 MMof VP-16 were added. Lane P without aclarubicin preincubation. Lanes Q to U, with increasing concentrations of aclarubicin (Lane Q, 10 nM; Lane R, 0.1 MM;Lane S, 1 MM;Lane T, 10 MM; 10 Lane U, 100 MMaclarubicin). c, coding strand; nc, noncoding strand.

alone had no stimulatory effect upon topoisomerase II-me diated DNA cleavage. The enzyme was then incubated with its DNA substrate for 5 min in the presence of varying concentrations of aclarubicin before addition of m-AMSA or VP-16. Incubation with m- 1 8 10 AMSA (10 ÃŸM)affected the site specificity of the enzyme in uM m-AMSA such a manner that cleavage was introduced at a number of Fig. 4. m-AMSA dose-response curves obtained on OC-NYH cells incubated for 60 min with ( ) and without ( ) preincubation for 20 min with 0.4 alternative DNA sequences (Fig. 6, Lane I). Preincubation with MMaclarubicin. Points, mean; bars, SE from triplicate cultures. aclarubicin at 10 MMor higher completely abolished topoisom- 3313

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. ACLARUBiriN INHIBITION OF VP-16 AND m-AMSA TOXKÃ•TY erase II cleavage (Fig. 6, Lanes M and N). Interestingly, in the 80 presence of only 10 nM aclarubicin, a synergistic action of m- <70 AMSA and aclarubicin was apparent (compare Lanes J and /), Q whereas 10 nM by itself had no effect upon topoisomerase II 260 cleavage (Lane D). Similar results were obtained when investi 050 gating the effect of aclarubicin on topoisomerase II-mediated o DNA cleavage in the presence of VP-16 (60 Â¿IM)(Fig.6, Lanes z 40 Q to U). Analogous to the results with m-AMSA, synergy Â§30 between very low concentrations of aclarubicin and VP-16 is Q. suggested (Fig. 6, Lane Q). This result was, however, not 20) O QÃ• corroborated in the clonogenic assay as even very low (3 nM) UJ . f. Q_ ' 0 aclarubicin concentrations were able to inhibit the toxicity of . -A & -A * VP-16 (Fig. 7) and m-AMSA (Fig. 8). To further elucidate the effect of aclarubicin observed in vitro, 100pM 1nM 10nM 100nM we investigated the influence of aclarubicin on m-AMSA- or 804 VP-16-stimulated, protein-concealed DNA strand breaks in cells. Consistent with the ability to enhance topoisomerase II- <70 B Q mediated DNA strand breaks with isolated enzyme, m-AMSA Seo and VP-16 produced protein-DNA cross-linked strand breaks 3 in cultured cells (Fig. 9). As shown in the figure approximately 050 o 60% and 80% of the total genomic DNA were found to be u 40!Â» --â€¢- protein concealed as measured after 30 min of incubation with either 10 Â¿IMm-AMSAor60/nM VP-16, respectively. Lowering the concentration to 1 /Â«Mm-AMSA and 6 /ÃŒMVP-16resulted i520 100.0 Â£10 A A A A r^^~2y p E 1nM lOnM 100nM 1Â¿jM R 100pM C ACLARUBICIN CONCENTRATION E N 10.0 Fig. 9. Effect of aclarubicin on drug-induced, protein-concealed DNA strand T breaks in cultured human Daudi cells. Cells were treated for 30 min with the indicated concentration of aclarubicin followed by treatment for 30 min with 10 MM m-AMSA (D) and I MM m-AMSA (*) (.-() or 60 MM VP-16 (O) and 6 MM VP- U R 16 (â€¢)(A). Aclarubicin alone for 60 min (A). The amount of protein-associated V DNA breaks was determined as described in "Materials and Methods." I 1.0 V A L in a decrease in protein-concealed DNA breaks. Further, the data presented in Fig. 9 demonstrate that aclarubicin inhibited 0.1 in a dose-dependent manner protein-associated DNA strand 0 5 10 15 20 25 JO 35 40 45 50 55 60 65 breaks produced by the topoisomerase II-targeted drugs. No nM ACLARUBICIN synergistic effect was observed when cells were incubated with Fig. 7. Effect of low aclarubicin concentrations on the cytotoxicity of VP-16. low concentrations of aclarubicin, as seen in the cleavage assay Dose-response curves obtained on OC-NYH cells incubated with increasing aclarubicin concentrations for 80 min without VP-16 ( ). prcineubalion for with naked DNA. 20 min with aclarubicin and 60-min incubation with both aclarubicin and VP-16 (10 MM VP-16. ; 20 UM VP-16. ). Points, mean; hors. SE from triplicate cultures. DISCUSSION A number of intercalating drugs including Adriamycin. dau- 100-1 norubicin, bisantrene, mitoxantrone, 2-methyl-9-hydroxyellip- ticinum, m-AMSA, and actinomycin D, all with antitumor activity, stimulate topoisomerase II-mediated formation of ÃŽ cleavable protein-DNA complexes (19). A biphasic response with stimulation of complex formation at low concentrations, 10. saturation, or inhibition at higher drug concentrations has been described both in vivo (20) and in vitro (6, 21). Ethidium bromide, on the other hand, intercalates into DNA without stimulation of topoisomerase-mediated cleavage (14) and also inhibits the low level of cleavable complex formation induced by the enzyme alone (15). Analogous to the results with ethidium bromide, we find that aclarubicin in vitro is able to inhibit 10 15 20 25 30 35 40 45 50 55 60 65 the baseline cleavage of naked DNA elicited by topoisomerase nM ACLARUBICIN II. Similarly we find that aclarubicin, over a wide range of Fig. 8. Effect of low aclarubicin concentrations on the cytotoxicity of m- concentrations, is able to inhibit the increased formation of AMSA. Dose-response cunes obtained on OC-NYH cells incubated with increas DNA cleavages induced by the classical topoisomerase II tar ing aclarubicin concentrations for 80 min without m-AMSA ( ), prcincubation for 20 min with aclarubicin and 60-min incubation with both aclarubicin and 5 geting drugs VP-16 and m-AMSA. At low aclarubicin concen jiM m-AMSA ( ). Pointa, mean; bars. SE from triplicate cultures. trations we observed synergy in the formation of cleavable 3314

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. ACLARUBICIN INHIBITION OF VP-16 AND m-AMSA TOXICITY complexes obtained with m-AMSA and VP-16 on naked DNA. rubicin, there is no or only modest reduction in the accumula In contrast we found that aclarubicin preincubation was able to tion of aclarubicin in anthracycline-resistant cells (26, 27). Thus completely inhibit the increase in the formation of protein- the lack of (or limited) cross-resistance to aclarubicin could concealed DNA breaks in cells treated with m-AMSA or VP- simply be explained by differences in drug accumulation. An 16. We do not know the reason for this discrepancy, but alternative explanation is that aclarubicin, in contrast to the linearized naked DNA interacting with only one protein prob topoisomerase targeting drugs, is independent of topoisomerase ably has a higher number of available binding sites, other drug levels for its cytotoxicity. This conclusion is supported by the and enzyme concentrations at target, and a different topology present study and is in accordance with previous results showing after intercalation than does DNA in living cells. The antago that aclarubicin cytotoxicity is independent of the proportion nism between aclarubicin and the topoisomerase II targeting of cells in the S phase (13), whereas the cytotoxicity of Adria drugs is substantiated by the striking effect of aclarubicin prein mycin, daunorubicin, mitoxantrone, and VP-16 is greatest in cubation on VP-16 and m-AMSA cytotoxicity in the clonogenic the S phase where topoisomerase levels are high (13, 28). assay. Ethidium bromide also inhibits the toxicity of VP-16 in The structural basis for anthracycline interactions with to cells (15), but at much higher concentrations (10 to 100 /Â¿M poisomerase II is not known. A recent report suggests that compared with 0.1 to 0.5 ^M for aclarubicin). With the short substituents at the sugar amino group can either inhibit DNA drug exposure times and low concentrations used in the present intercalation or topoisomerase II targeting activity, depending investigation, the toxicity of aclarubicin is very limited, and the on the nature of the substituent (29). Thus, the double methyl- drug seems to be a good alternative to ethidium bromide when ation of the amino group in aclarubicin (Fig. 10) could be the investigating the role of topoisomerase-mediated drug toxicity modification responsible for the shift from stimulation to in in cell culture systems. hibition of topoisomerase II-mediated DNA cleavage. Resistance to anthracyclines has been attributed to a reduced The results of this study suggest that simultaneous adminis cellular drug accumulation (22). However, there are several tration of aclarubicin and VP-16 to patients is unlikely to be reports indicating that a reduced topoisomerase II level could advantageous even though the two drugs presumably have a also play a role (7, 23-25). Limited cross-resistance between low degree of cross-resistance. However, a recent Phase II trial aclarubicin and the "classical" anthracyclines Adriamycin and of VP-16 and aclarubicin administered simultaneously to pa daunorubicin has been described in a number of anthracycline- tients with relapse of acute myelocytic leukemia resulted in a resistant cell lines (9-12). Contrary to Adriamycin and dauno- complete remission rate of 40% (2). A possible explanation for this finding is that aclarubicin alone was sufficient and VP-16 thus wasted on these patients. In favor of this view is the high ACLARUBICIN response rate obtained in acute myelocytic leukemia with acla rubicin alone (70% complete remission in previously untreated COOCH, patients) (1). However, aclarubicin is converted to active and CH2CH3 inactive metabolites in vivo (30), and the extent to which the OH metabolites are responsible for the antitumor effect observed in vivo is not known. We are currently addressing the question of aclarubicin and VP-16 scheduling in vivo using murine tumor models in order to elucidate this matter of potential clinical importance.

ACKNOWLEDGMENTS We wish to thank Dr. Ole Westergaard for helpful discussions, Eva H0j, Annette Nielsen, and Birgit DalÃforexcellent technical assistance, CH, and John Post for preparing the manuscript.

REFERENCES

1. Hansen. O. P., Ellegaard, J.. Madsen. P. B., Brincker, H., Christensen, B. E., Killmann, S. A., Laursen, M. L., Karle. H.. Drivsholm, A.. Jensen. M. K., Laursen, B.. Jans. H., Hippe, E., Pedersen-Bjergaard, J., Nissen, N., Thorling, K., and Jensen. K. B. Combination chemotherapy with aclarubicin ADRIAMYCIN plus cytosine arabinoside versus daunorubicin plus cytosine arabinoside in de nom acute myelocytic leukemia (AML): a Danish national trial. Proc. Am. Soc. Clin. Oncol.. 7: 175. 1988. O OH 2. Rowe, J. M., Chang, A. Y. C., and Bennett, J. M. Aclacinomycin A and etoposide (VP16-213). An effective regimen in previously treated patients COCH2OH with refractory acute myelogenous leukemia. Blood, 71: 992-996, 1988. OH 3. Pedersen-Bjergaard, J.. Brincker, H., Ellegaard. J.. Drivsholm, A., Freund, L.. Jensen. K. B.. Jensen, M. K., and Nissen, N. I. Aclarubicin in the treatment of acute nonlymphocytic leukemia refractory to treatment with daunomycin and cytarabine: a Phase II trial. Cancer Treat. Rep., 68: 1233- 1238, 1984. 4. Nelson, E. M., Tewey, K. M.. and Liu, L. L. Mechanism of antitumor drug action: poisoning of mammalian DNA topoisomerase II on DNA by 4'- (acridinylamino)methanesulfon-m-anisidide. Proc. Nati. Acad. Sci. USA. 81: 1361-1365. 1984. 5. Yang, L., Rowe, T. C., and Liu, L. L. Identification of DNA topoisomerase II as intracellular target of antitumor epipodophyllotoxines in simian virus 40-infected monkey cells. Cancer Res., 45: 5872-5876, 1985. 6. Tewey. K. M., Rowe, T. C., Yang. L., Halligan, B. D.. and Liu, L. F. Fig. 10. Structures of aclarubicin and Adriamycin. Adriamycin-induced DNA damage mediated by mammalian DNA topoisom- 3315

erase II. Science (Wash. DC). 226: 466-468. 1984. double-stranded DNA cleavage reaction. Biochemistry. 28:6237-6244. 1989. 7. Deffie, A. M., Batra. J. K.. and Goldenberg, G. J. Direct correlation between 19. Bodley. A. L., and Liu. L. F. Roles of DNA topoisomerases in drug cytotox DNA topoisomerase II activity and cytotoxicity in Adriamycin-sensitive and icity and drug resistance. In: P. V. Wooley and K. D. Tew (eds.). Mechanisms -resistant P388 leukemia cell lines. Cancer Res., 49: 58-62, 1989. of Drug Resistance in Neoplastic Cells, pp. 277-286. San Diego: Academic 8. DuVerney, V. H., Pachter, J. A., and Crooke, S. T. Deoxyribonucleic acid Press, 1988. binding studies on several new anthracycline antitumor antibiotics. Sequence 20. Finlay, G. J., and Baguley, B. C. Selectivity of yV-[2-(dimethylamino)clhyl] preference and structure-activity relationships of marcellomycin and its an acridine-4-carboxamide towards Lewis lung carcinoma and human tumor alogues as compared to Adriamycin. Biochemistry, IS: 4024-4030, 1979. cell lines in vitro. Eur. J. Cancer Clin. Oncol., 25: 271-277, 1989. 9. Scott, C. A., Westmacott. D., Broadhurst, M. J.. Thomas, G. J., and Hall, 21. Pommier, Y., Minford, J. K., Schwartz. R. E., dwelling, L. A., and Kohn, M. J. 9-Alkyl anthracyclines. Absence of cross-resistance to Adriamycin in K. W. Effects of the DNA intercalators4'-(9-acridinylamino)niethanesulfon- human and murine cell cultures. Br. J. Cancer, 53: 595-600, 1986. m-anisidide and 2-methyl-9-hydroxycllipticinium on topoisomerase II me 10. Hill, B. T., Dennis, L. Y., Li, X. T., and Whelan, R. D. H. Identification of diated DNA strand cleavage and strand passage. Biochemistry. 24: 6410- anthracycline analogues with enhanced cytotoxicity and lack of cross-resist 6416. 1985. ance to Adriamycin using series of mammalian cell lines in vitro. Cancer 22. Dane. K. Active outward transport of daunomycin in resistant Ehrlich ascites Chemother. Pharmacol.. 14: 194-201, 1985. tumor cells. Biochim. Biophys. Acta, 323: 466-483, 1973. 11. Twentyman. P. R., Fox, N. E., Wright. K. A., and Bleehen. N. M. Derivation 23. Glisson, B.. Gupta, R., Hodges, P., and Ross, W. Cross-resistance to inter and preliminary characterization of Adriamycin resistant lines of human lung calating agents in an epipodophyllotoxin-resistant Chinese hamster ovary cancer cells. Br. J. Cancer, S3: 529-537. 1986. cell line: evidence for a common intracellular target. Cancer Res.. 46: 1939- 12. Jensen. P. B., Vindelav. L., Roed. H.. Demant, E. J. F., Sehested, M., 1942. 1986. Skovsgaard, T., and Hansen. H. H. In vitro evaluation of the potential of 24. Potmesil, M.. Hsiang. Y-H., Liu, L. F., Wu, H-Y., TrÃ¡ganos. F.. Bank. B., aclarubicin in the treatment of small cell carcinoma of the lung (SCCL). Br. and Silber. R. DNA topoisomerase II as a potential factor in drug resistance J. Cancer, 60: 838-844, 1989. of human malignancies. Proceedings of the conference on DNA topoisomer 13. Jensen. P. B., Roed. H.. Vindel0v. L.. Christensen. I. J.. and Hansen. H. H. ases in cancer chemotherapy. NCI Monogr.. 4: 105-109, 1987. Reduced variation in the clonogenic assay obtained by standardization of the 25. Davies, S. M., Robson, C. N.. Davies. S. L., and Hickson, I. D. Nuclear cell culture conditions prior to drug testing on human small cell lung cancer topoisomerase If levels correlate with the sensitivity of mammalian cells to cell lines. Invest. New Drugs. 7: 307-315. 1989. intercalating agents and epipodophyllotoxins. J. Biol. Chem., 263: 17724- 14. Tewey, K. M., Chen, G. L., Nelson, E. M.. and Liu, L. F. Intercalate 17729, 1988. antitumor drugs interfere with the breakage-reunion reaction of mammalian 26. Skovsgaard, T. Pharmacodynamic aspects of aclarubicin with special refer DNA topoisomerase II. J. Biol. Chem.. 259: 9182-9187, 1984. ence to daunorubicin and doxorubicin. Eur. J. Haematol.. 38: 7-20. 1987. 15. Rowe, T., Kupfer, G., and Ross. W. Inhibition of epipodophyllotoxin cyto- 27. Seeber. S., Loth. H.. and Crooke, S. T. Comparative nuclear and cellular toxicity by interference with topoisomerase-mediated DNA cleavage. incorporation of daunorubicin, doxorubicin, carminomycin, marcellomycin, Biochem. Pharmacol.. 34: 2483-2487, 1985. aclacinomycin A. and AD 32 in daunorubicin-sensitive and -resistant Ehrlich 16. Roed, H.. Christensen. I. J.. Vindclov, L. L.. Spang-Thomsen, M., and ascites in vitro. J. Cancer Res. Clin. Oncol., 98: 109-118, 1980. Hansen. H. H. Inter-experiment variation and dependence on culture con- 28. Chow, K., and Ross, W. Topoisomerase-specific drug sensitivity in relation ditions in assaying chemosensitivity of human small cell lung cancer lines. to cell cycle progression. Mol. Cell. Biol., 7: 3119-3123, 1987. Eur. J. Cancer Clin. Oncol., 23: 177-186, 1987. 29. Bodley, A., Liu, L. F.. Israel. M., Seshadri. R.. Koseki. Y.. Giuliani, F. C., 17. Allen, L. M. Comparison of uptake and binding of two epipodophyllotoxin Kirschenbaum, R., Silber, R., and Potmesil, M. DNA topoisomerase II- glucopyranosides, 4'-demethyl epipodophyllotoxin thenylidene-fi-D-gluco- mediated interaction of doxorubicin and daunorubicin congeners with DNA. side and 4'-demethyl epipodophyllotoxin ethylidene-rf-n-glucoside. in the Cancer Res., 49: 5969-5978. 1989. L1210 leukemia cell. Cancer Res., 3S: 2549-2554. 1978. 30. Komiyama. T.. Oki. T.. Inui. T.. Takeuchi. T., and Umezawa. H. Reduction 18. Andersen. H. A., Christiansen. K., Zechiedrich, E. L., Jensen, P. S.,Osheroff. of cinerulose in aclacinomycin-A by soluble and microsomal cinerulose N., and W'estergaard, O. Strand specificity of the topoisomerase II mediated reductases. Gann. 70: 395-401. 1979.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. Antagonistic Effect of Aclarubicin on the Cytotoxicity of Etoposide and 4 ′-(9-Acridinylamino)methanesulfon-m-anisidide in Human Small Cell Lung Cancer Cell Lines and on Topoisomerase II-mediated DNA Cleavage

Peter Buhl Jensen, Boe Sandahl Sørensen, Erland J. F. Demant, et al.

Cancer Res 1990;50:3311-3316.

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