Molecular Effects of Topoisomerase II Inhibitors in AML Cell Lines

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Leukemia (1999) 13, 1859–1863  1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu Molecular effects of topoisomerase II inhibitors in AML cell lines: correlation of apoptosis with topoisomerase II activity but not with DNA damage F Gieseler1, E Bauer2, V Nuessler3, M Clark1 and S Valsamas2

1University Hospital, Klinik fu¨r Allgemeine Innere Medizin, Kiel; 2University Hospital, Medizinische Poliklinik, Wu¨rzburg, Germany; and 3University Hospital, Klinikum Grosshadern, Munich, Germany

We examined the cellular effects of topo II inhibitors in two not consistent with this model. Resistant cells ultimately human myeloid cell lines, HL-60 and KG-1 cells, with the pur- develop mechanisms that allow for surviving DNA damage pose of finding molecular markers for the sensitivity of leuke- 4–6 mia cells to topo II inhibitors. These cell lines are widely used, induced by anthracyclines. In addition to DNA damage, well characterized and they differ in their sensitivities to topo other apoptosis mechanisms such as the CD95/FAS-pathway II inhibitors. Despite the fact that HL-60 cells are p53-negative, seem to be important. Friesen et al7 have shown that blockage they are much more sensitive than KG-1 cells. Three different of the CD95-receptor inhibits daunorubicin-induced topo II inhibitors with distinct molecular ways of action have apoptosis. Aclarubicin, on the other hand, is also able to been used. Daunorubicin and aclarubicin are DNA intercalators induce apoptosis although no topo II/DNA complex is formed. that secondarily interact with topo II; etoposide, on the other hand, directly binds to the enzyme. In contrast to daunorubicin, In contrast to the classical anthracyclines such as daunorub- which induces protein-associated DNA double-strand breaks icin or doxorubicin, aclarubicin inhibits the access of topo II due to the blockage of topo II action, aclarubicin inhibits the to the DNA. A number of clinical studies have proven the access of DNA by topo II. No correlation could be established effectiveness of aclarubicin in the therapy of AML.8–12 between the drug-induced DNA damage and apoptosis. In fact, Several different apoptosis pathways, including the acti- the amount and pattern of DNA damage examined with the vation of p53, involvement of the CD-95/FAS-pathway, tumor ‘comet assay’ was characteristic for each drug in both cell necrosis factor or cytochrome-C release by mitochondria, lines. The DNA binding of daunorubicin was slightly higher in 13 HL-60 cells, but there was no notable variance between the cell have been proposed. Obviously, the pathways that are util- lines for aclarubicin. The most striking difference could be ized are specific for different cell types. The induction of the found for the nuclear topo II activity, which was about half in CD-95/FAS-system by daunorubicin for example, seems to be KG-1 cells and, additionally, less than 1% of the nuclear topo a preferred mechanism in lymphatic cells, but not in myelo- II activity was bound to the DNA in KG-1 cells when compared blastic leukaemia cells.7,14,15 to HL-60 cells. This fraction of topo II interacts with the inhibitors; subsequently these findings might well explain the vari- The development of cellular resistance is a major problem ance in the cellular sensitivity. Additional factors are alter- in the therapy of AML. The elaboration of therapeutic stra- ations of the apoptotic pathways, eg loss of p53 in HL-60 cells. tegies to recognize and overcome cellular resistance requires Although we found no differences in the quantity of DNA dam- the knowledge of the molecular determinants for response to age between the cell lines after drug treatment, the quality of the therapy with topo II inhibtors. In this paper, the intracellu- DNA damage appeared to be distinct for each topo II inhibitor. lar effects of etoposide, daunorubicin and aclarubicin have The morphological appearance of the comet tails after treatment was characteristic for each drug. Further studies are been examined using two different human myeloblastic cell necessary to decide whether these in vitro data are compatible lines. HL-60 and KG-1 cells were selected because they are with the clinical situation. widely used and well-characterized cell lines, and they exhi- Keywords: chemotherapy, leukemia, topoisomerase, DNA dam- bit major differences in their sensitivity to topo II inhibitors. age

Materials and methods Introduction Cell lines Topoisomerase II (topo II) inhibitors are the backbone of most hemato-oncological strategies, although the molecular events that result in apoptotic cell death are still unveiled. Anthracy- HL-60 cells (ATCC 45500) were cultured in RPMI 1640, clines and epipodophyllotoxines use different ways to inhibit 25 mM HEPES (Sigma, St Louis, MO, USA) with 2 mML-gluta- the DNA processing enzyme topo II; whereas anthracyclines mine, penicillin/streptamycin, and 7.5% FCS (Gibco, Grand are intercalators and secondarily interact with topo II, epipo- Island, NY, USA). KG-1 cells (ATCC CRL-8031) were cultured dophyllotoxines bind directly to the enzyme.1,2 Both types of in Iscove’s modiﬁed Dulbecco’s medium (Sigma) with 2 mM inhibitors block the action of topo II after the induction of L-glutamine, 20% FCS (Gibco). Cultures were incubated in the DNA double-strand breaks. This results in DNA damage with presence of 5% CO2 in a water-jacketed incubator (Forma covalent binding of topo II to the DNA ends.3 It is widely Scientiﬁc, Germany). accepted that the combination of protein-associated DNA damage with the loss of the enzyme’s activity ultimately induces cell death. However, a number of observations are Topoisomerase II inhibitors

Drug concentrations were the same for all experiments: etopo- Correspondence: F Gieseler, Klinik fu¨r Allgemeine Innere Medizin side (Bristol-Myers Squibb, Munich, Germany) 1 ␮g/ml; dau- der Christian-Albrechts-Universita¨t, Schwerpunkt Ha¨matologie/ Onkologie, Schittenhelmstrasse 12, 24105 Kiel, Germany; Fax: +431 norubicin (Pharmacia and Upjohn, Erlangen, Germany) 597 1302 0.1 ␮g/ml; aclarubicin (MEDAC, Hamburg, Germany) Received 12 August 1998; accepted 12 July 1999 0.1 ␮g/ml. Due to the high protein-binding of anthracyclines Effects of topoisomerase II inhibitors in AML cell lines F Gieseler et al 1860 it is mandatory to use the same extracellular protein concen- and 75 ␮l 1% low melting point agarose for the upper layer. trations for toxicological tests. The agarose slides are then lysed in 1% Triton X-100, 10% DMSO (Sigma) and 89% lysis buffer, pH 10 (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% N-laurylsarcosine sodium salt Alamar blue assay for at least 1 h. The agarose slides are equilibrated for 20 min in electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH 200 ␮l cell suspension with 2.5 × 105 cells/ml were incubated Ͼ12.5) and subsequently the electrophoresis is run at 25 volts at 37°C for in the presence of drugs for 72 h on 96-well flat- and 300 milliampere for 20 min. The slides were neutralized bottom tissue culture plates with low evaporation lids (Costar, in several changes of 0.4 M Tris, pH 7.5 and the DNA stained Bodenheim, Germany). Then 25 ␮l/well Alamar blue with 50 ␮lof20␮g/ml ethidium bromide solution. The cells (BioSource, Ratingen, Germany) was added and the cells were catalogued using a fluorescence microscope and photo- incubated for an additional 18 h at 37°C. The fluorescence graphed. Cells with DNA tails longer than one diameter of the was subsequently measured on a LS50B luminescence spec- nucleus were calculated as positive for DNA damage. trometer (Perkin Elmer, Konstanz, Germany) with excitation at

540 nM and emission at 590 nM. The IC50 and IC90 concentrations were calculated from the arbitrary units using the con- Topoisomerase II activity trol measurements for 0% proliferation (medium without cells) and 100% (cell suspension without drugs). 2.5 × 105 cells/ml were incubated with the desired drug concentrations for 3 h. Subsequently the nuclei were isolated on ice with 8 ml lysis buffer (0.3 M sucrose; 0.5 mM EGTA, pH DNA binding of drugs in intact cells 8.0; 60 mM KCl; 15 mM NaCl; 15 mM HEPES, pH 7.5; 150 ␮M spermidine (Sigma) +40 ␮l Triton X-100). The nuclei were Two ml cell suspension with 2.5 × 105 cells/ml were incu- counted and washed in lysis buffer without Triton X-100. The bated at 37°C for 30 min in HBSS containing 6.25 ␮g/ml nuclei pellet was re-suspended in extraction buffer (5 mM K- Hoechst dye 33342 (Hoechst, Bad Soden, Germany). After Phosphate, pH 7.5; 100 mM NaCl (Roth); 1 ␮l/ml 2-mercato- addition of drugs, the cells were incubated at 37°C for an ethanol; 5 ␮l/ml 200 mM PMSF (Sigma) at a concentration of additional 30 min. Quenching of Hoechst-dye fluorescence 3 × 107 nuclei/ml. 90 ␮l5M NaCl/ml extraction buffer was was subsequently measured using a Partec flow-cytometer. added (350 mM) and the tube mixed by inverting. After 15 min on ice, the DNA and undissolved protein was pelleted by cen- trifugation, 12 000 g for 10 min. The DNA/protein pellet was Apo-2.7 detection removed from the extract to a new tube and the same amount of extraction buffer was added as in the previous step. Re- Cells were incubated with drugs for 1 h, then washed and extraction to determine the fraction of DNA-bound topoiso- resuspended in complete medium for 24 h. 5 × 105 cells then merase activity was then done with 900 mM NaCl (200 ␮l/ml were washed with PBS and permeabilized with 100 ␮l ice extraction buffer). Both first and second extracts were stored cold digitonin (100 ␮g/ml) dissolved in PBS supplemented at −20°C in 50% glycerol.

with 2.5% FCS and 0.01% NaN3 for 20 min on ice. After Topoisomerase II activity was quantiﬁed using a modiﬁ- washing the pellet, 10 ␮l of the MoAb Apo2.7-PE (Coulter cation of a standard relaxation assay as published before:16,17 Immunotech, Marseille, France) or an isotype-matched con- 1 ␮g pBR 322 supercoiled plasmid DNA was relaxed with trol were added and gently mixed. Cells were incubated for nuclear extracts using assay buffers (0.5 M bis-tris-propane;

15 min at room temperature in the dark, washed and analyzed 900 mML-glutamic acid, monopotassium salt; 5 mM MgCl2. on a FACScan flow cytometer with Cell Quest software 75 ␮g/ml BSA; 10 mM ATP, 50 mM DTT) with an adjusted pH (Becton Dickenson, Heidelberg, Germany). of 8.9. Topo I activity was inhibited by the addition of 1 ␮M camptothecin. The gels were stained with ethidium bromide and photographed with Polaroid 667 black and white film. CD95 expression The photos were scanned with an Apple One Scanner and a grey-scale analysis performed. One unit is defined as being 5 × 105 cells were washed with PBS, then stained for CD95 the amount of activity from 1 × 104 nuclei required to relax with 10 ␮l of the monoclonal antibody anti-CD95 UB2 90% of 500 ng DNA. (Coulter Immunotech) or a control IgG-1 for 15 min at 4°Cin the dark. Subsequently, the cells were washed and analyzed on a FACScan flow cytometer with Cell Quest software Results (Becton Dickenson). In this paper, the cellular effects of topoisomerase II inhibitors in two different cell lines have been compared. HL-60 cells Drug-induced DNA damage and KG-1 cells are widely used human myeloblastic cell lines. Some characteristics of these cells are shown in Table 1. HL- The single cell microgel electrophoresis assay (‘comet 60 cells had a doubling time of 22.2 h; KG-1 cells in contrast assay’): Cells were cultured for 3 h with or without drugs, needed 64.1 h to duplicate as displayed in Table 1. then centrifuged and re-suspended in fresh medium without drugs and incubated for an additional 21 h at 37°C before being prepared for electrophoresis. 5 × 104 cells in 75 ␮l1% DNA binding of drugs and induction of apoptosis low melting point agarose (Sigma) were sandwiched between two layers of agarose, 85 ␮l 0.5% NEEO agarose (Roth, In contradiction to the loss of p53, HL-60 cells are much more Deisenhofen, Germany) for the foundation layer on the slide sensitive to topoisomerase II inhibitors as KG-1 cells (Table 2). Effects of topoisomerase II inhibitors in AML cell lines F Gieseler et al 1861 Table 1 Some characteristics of HL-60 and KG-1 cells Table 5 Free and DNA-bound topo II activity in nuclear extracts

Characteristics HL-60 cells KG-1 cells Fraction HL-60 cells KG-1 cells

Origin human, acute human, acute Total (U)a 32.73 (±3.14) 15.65 (±3.15) myelogenous myelogenous Free (% of total) 82.48 (±7.10) 99.62 (±19.45) leukaemia (ATCC leukaemia (ATCC DNA-bound (% of 17.53 (±2.52) 0.32 (±0.68) 45500) CRL-8031) total) Doubling time 22.2 ± 3.5 64.1 ± 15.2 ± (h s.d.) aUnit deﬁnition as in Materials and methods, median of three experi- 30,31 p53 status gene deletion inducible gene ments (±s.d.) expression

Table 2 Sensitivity of HL-60 cells and KG-1 cells (␮g/ml)

HL-60 cells KG-1 cells

± ± Etoposide IC50 0.156 ( 0.07) 31.57 ( 12.5) ± ± IC90 0.372 ( 0.01) 186.1 ( 1.43) Daunorubicin IC50 0.004 (±0.003) 0.958 (±4.60) ± ± IC90 0.822 ( 0.04) 23.13 ( 1.75) ± ± Aclarubicin IC50 0.0004 ( 0.003) 18.61 ( 14.4) IC90 0.017 (±0.01) 31.57 (±12.5) Figure 1 Free and DNA-bound topo II activity in nuclear extracts 21 h after a 3-h treatment shown as percentage of total activity ± Continuous incubation for 72 h, median of three experiments ±s.d. (median of three experiments, s.d.). ctr, control; eto, etoposide; dau, daunorubicin; acla, aclarubicin.

Topo II activity before and after treatment Table 3 Induction of apo-2.7 by etoposide and aclarubicin (% positive cells) The topo II activity in the nuclei of KG-1 cells was almost half the activity in HL-60 nuclei (Table 5). Moreover, only 0.32% Drug HL-60 cells KG-1 cells of the total topo II activity in KG-1 cells was DNA-bound (Table 5). After incubation of the cells with topo II inhibitors, ± ± Control 21.59 ( 1.16) 63.69 ( 4.80) the shift in the relation between free and DNA-bound topo II Etoposide 49.09 (±1.25) 94.47 (±0.54) Aclarubicin 79.43 (±0.15) 87.09 (±0.87) activity has been measured. As shown in Figure 1, etoposide as well as daunorubicin increased the relation of DNA-bound Incubation for 1 h, measurement after 23 h; median of three experi- to free activity. Incubation of the cells with aclarubicin in con- ments (±s.d.). trast, reduced the DNA-bound fraction in HL-60 cells and resulted in only a slight increase in KG-1 cells. For the interpretation of the results, the lower total activity and the low fraction of DNA-bound activity in untreated KG-1 cells Table 4 DNA-binding rate of topoisomerase II inhibitors must be taken into consideration.

Drug HL-60 cells KG-1 cells Nuclear swelling and DNA damage after treatment Etoposide (1.0 ␮g) −20.59 (±4.72) −1.18 (±4.27) Daunorubicin (0.1 ␮g) 84.57 (±8.71) 69.09 (±5.95) ␮ ± ± It has been suggested that the DNA damage after treatment Aclarubicin (0.1 g) 93.55 ( 2.15) 95.65 ( 0.6) with topo II inhibitors is an important factor for the induction of apoptosis.18–20 We did not ﬁnd notable differences between Quenching of Hoechst 33342 ﬂuorescence in % to control, median the sensitive HL-60 cells and the more resistant KG-1 cells of three experiments (±s.d.). (Figure 2), 21 h after a 3-h treatment. As well as daunorubicin,

In addition to the alamar blue assay which has been used for the experiments presented in Table 2, cell death had been estimated by trypan blue exclusion, with very similar data. Alterations of the mitochondrial membrane that are characteristic for apoptotic cell death, were detected by the appearance of Apo-2.7 (Table 3). Due to its high fluorescence, daunorubicin could not be analyzed in this setting. In both cell types, daunorubicin as well as aclarubicin had high DNA binding rates (Table 4). The DNA binding rate of daunorubicin was Figure 2 DNA damage 21 h after a 3-h treatment shown as a frac- considerably higher in HL-60 cells than in KG-1 cells. We did tion of cells with comets longer than the diameter of the nucleus (%), not find these differences for aclarubicin (Table 4). median of three to four experiments, ±s.d.). Abbreviations as Figure 1. Effects of topoisomerase II inhibitors in AML cell lines F Gieseler et al 1862 etoposide induced DNA damage in more than 80% of the tivity of these cells as we have shown previously that a posi- cells. The number of positive cells after treatment with aclaru- tive and linear correlation between the DNA binding of bicin was considerably lower. One advantage of the comet daunorubicin and the rate of apoptosis exists.1 This is not the assay is the possibility of examining the nuclei and the case for aclarubicin where the binding was more than 90% resulting comet tails morphologically. In Figure 3, the typical in both cell lines. appearance of comets 21 h after a 3-hour treatment is shown. The decisive differences between the cell lines were found After incubation with aclarubicin, the nuclei were significantly within the topo II activities. The total topo II activity in nuclear larger than after treatment with etoposide or daunorubicin extracts could be separated in a free and a DNA-bound por- (diameter of nuclei after etoposide, 0.74 ± 0.08 ␮m; daunoru- tion.21,22 In order to be active and process DNA, the enzyme bicin 0.68 ± 0.1 ␮m; aclarubicin 2.94 ± 0.20 ␮m). Moreover, must bind to the DNA.20,23,24 As shown in Table 5, the total the patterns of the comets are distinct for the different drugs; topo II activity of KG-1 cells is about 50% of the activity of the comet tails after aclarubicin treatment are much more HL-60 cells. Moreover, only 0.32% of the total activity is granular, than the comet tails after etoposide or dauno- bound to the DNA in KG-1 cells. As this is the only topoiso- rubicin treatment. merases portion that can be effectively inhibited by the drugs, None of the topo II inhibitors increased the expression of these variances might well explain the difference in the sensi- the CD95 receptor more than 20% as compared to the con- tivity of the cells to topo II inhibitors. One of the reasons for trol, which is consistent with the literature (McGahon).32 the higher topo II activity in HL-60 cells is their higher proliferation rate with a doubling time of 22.2 h compared to 64.1 h for KG-1 cells (Table 1). Discussion At least two reasons can be discussed for the discrepancy of lower topo II activity in untreated KG-1 cells with similar In this paper, the molecular effects of the topo II inhibitors DNA damage after treatment. One of them is the accumu- etoposide, daunorubicin and aclarubicin in a sensitive and a lation of DNA strand breaks during drug treatment. With the resistant human myeloid cell line have been examined. The aim to monitor the trapping of topo II to the DNA by the drugs most intriguing result is that we did not find notable differ- during the treatment period, we performed the experiments ences in the DNA damage 24 h after treatment with the same shown in Figure 1. Drug treatment results in a considerable drug concentrations, although HL-60 cells are much more shift from the free topo II fraction towards the DNA-bound sensitive than KG-1 cells (Figure 2). In addition, aclarubicin, fraction. This is true for both cell lines, although to a higher which causes considerably less DNA damage than daunorub- extent for daunorubicin in HL-60 cells. The larger DNA bound icin or etoposide, is also a strong initiator of apoptosis. It fraction after treatment of KG-1 cells with aclarubicin which seems that the drug-induced DNA damage does not correlate does not induce the formation of a ‘cleavable complex’, might with apoptosis. reflect an unspecific activation of topo II by drug treatment. In Table 4, the DNA binding rate of topo II inhibitors in A second reason is the sensitivity of the single cell microgel intact HL-60 cells and KG-1 cells is shown. As expected, eto- electrophoresis assay (‘comet assay’) which has been used to poside does not bind to DNA. Daunorubicin and aclarubicin quantify the DNA damage. This assay allows the combination are both strong intercalators with high DNA binding rates. The of morphological examination of the nuclei and the quantifi- DNA binding of daunorubicin was 15.48% higher in the more cation of DNA damage. Under the conditions we used, with sensitive HL-60 cells. We did not find these differences for a pH of the clectrophoresis buffer Ͼ12.5, DNA single-strand aclarubicin. The higher DNA binding of daunorubicin in HL- breaks, as well as DNA double-strand breaks are detected.25,26 60 cells could well be one of the factors for the higher sensi- In contrast to the epipodophyllotoxins, anthracyclines are able to induce DNA damage in several ways in addition to the inhibition of topo II. One of them is the intracellular pro- duction of free oxygen radicals, which had been held respon- sible for the cumulative cardiotoxicity of anthracyclines.27–29 Although we did not examine the nature of DNA damage in this project (eg single- or double-strand breaks, protein associated or not, etc), the composition of DNA damage induced by the different drugs as displayed in Figure 2 are distinct. Classical anthracyclines, such as daunorubicin, induce both topo II associated DNA damage and damage due to free oxygen radicals. Aclarubicin also produces free oxygen radicals but its action does not result in protein associated DNA damage. Etoposide does not produce free oxygen radicals and all DNA damage should arise from topo II inhibition. The morphological appearance of the nuclei and their comet tails are distinct for the various drugs (Figure 3). This might be explained by their different modes of intracellular action. Etoposide and daunorubicin induce small apoptotic nuclei with long tails. Treatment with aclarubicin results in a swelling of the nuclei and a more granular pattern of the DNA tail. This might be explained by the inhibition of topo II action Figure 3 Microscopical appearance of comets 21 h after a 3-h without the formation of a ‘cleavable complex’ by aclarubicin treatment of HL-60 cells. (A) Control; (B) etoposide; (C) daunorubicin; (D) aclarubicin. Diameters of nuclei after treatment: etoposide, that results in the inability of the cell to supercoil DNA. 0.74 ± 0.08 ␮m; daunorubicin 0.68 ± 0.1 ␮m; aclarubicin The knowledge of intracellular effects and specific apop- 2.94 ± 0.20 ␮m (median of three experiments ±s.d.). totic signals of topo II inhibitors is mandatory for the future Effects of topoisomerase II inhibitors in AML cell lines F Gieseler et al 1863 design of efficacious therapeutic strategies. Evidently, the 14 Iijima N, Miyamura K, Itou T, Tanimoto M, Sobue R, Saito H. nuclear topo II activity is a suitable marker for the cellular Functional expression of Fas (CD95) in acute myeloid leukemia sensitivity to topo II inhibitors. Further studies are necessary cells in the context of CD34 and CD38 expression: possible correlation with sensitivity to chemotherapy. Blood 1997; 90: 4901– to decide whether these in vitro data are compatible with the 4909. clinical situation. 15 Eischen CM, Kottke TJ, Martins LM, Basi GS, Tung JS, Earnshaw WC, Leibson PJ, Kaufmann SH. Comparison of apoptosis in wild- type and Fas-resistant cells: chemotherapy-induced apoptosis is Acknowledgements not dependent on Fas/Fas ligand interactions. Blood 1997; 90: 935–943. 16 Clark M, Gieseler F. A method for the determination of topoiso- This work has been supported by Sander Stiftung (grant merase II alpha and beta activity in patient cells. Proc AACR 1994; 96.052.1) and Deutsche Forschungsgemeinschaft (SFB 172, 34: 209. C9). 17 Gieseler F, Glasmacher A, Kampfe D, Wandt H, Nuessler V, Val- samas S, Kunze J, Wilms K. Topoisomerase II activities in AML and their correlation with cellular sensitivity to anthracyclines and epipodophyllotoxines. Leukemia 1996; 10: 1177–1180. References 18 Solary E, Bertrand R, Pommier Y. Apoptosis induced by DNA topoisomerase I and II inhibitors in human leukemic HL-60 cells. 1 D’Incalci M. DNA-topoisomerase inhibitors. Curr Opin Oncol Leuk Lymphoma 1994; 15: 21–32. 1993; 5: 1023–1028. 19 Nelson WG, Kastan MB. DNA strand breaks: the DNA template 2 Burden DA, Kingma PS, Froelich Ammon SJ, Bjornsti MA, Patchan alterations that trigger p53-dependent DNA damage response MW, Thompson RB, Osheroff N. Topoisomerase II etoposide inter- pathways. Mol Cell Biol 1994; 14: 1815–1823. actions direct the formation of drug-induced enzyme–DNA cleav- 20 Gieseler F, Nu¨βler V, Brieden T, Kunze J, Valsamas S. Intracellular age complexes. J Biol Chem 1996; 271: 29238–29244. pharmacokinetics of anthracyclines in human leukemia cells: cor- 3 Tewey KM, Rowe TC, Yang L, Halligen BD, Liu LF. Adriamycin- relation of DNA-binding with apoptotic cell death. Int J Pharmacol induced DNA damage mediated by mammalian DNA topoiso- 1998; 36: 25–28. merase II. Science 1984; 226: 466–468. 21 Danks MK, Qiu J, Catapano CV, Schmidt CA, Beck WT, Fernandes 4 Nishiyama M, Horichi N, Mazouzi Z, Bungo M, Saijo N, Tapiero DJ. Subcellular distribution of the alpha and beta topoisomerase H. Can cytotoxic activity of anthracyclines be related to DNA II–DNA complexes stabilized by VM-26. Biochem Pharmacol damage? Anticancer Drug Des 1990; 5: 135–139. 1994; 48: 1785–1795. 5 Belvedere G, Suarato A, Geroni C, Giuliani FC, D’Incalci M. Com- 22 Fernandes DJ, Danks MK, Beek WT. Decreased nuclear matrix parison of intracellular drug retention, DNA damage and cytotox- DNA topoisomerase II in human leukemia cells resistant to VM- icity of derivatives of doxorubicin and daunorubicin in a human 26 and m-AMSA. Biochemistry 1990; 29: 4235–4241. colon adenocarcinoma cell line (LoVo). Biochem Pharmacol 23 Osheroff N. Biochemical basis for the interactions of type I and 1989; 38: 3713–3721. type II topoisomerases with DNA. Pharmacol Ther 1989; 41: 6 de Tinguy-Moreaud E, Pourquier P, Montaudon D, Robert J. 223–241. Relationships between DNA damage and growth inhibition 24 Robinson MJ, Osheroff N. Effects of antioncoplastic drugs on the induced by topoisomerase II-interfering drugs in doxorubicin- post-strand-passage DNA cleavage/religation equilibrium of topo- sensitive and -resistant rat glioblastoma cells. Anticancer Res isomerase II. Biochem 1991; 30: 1807–1831. 1994; 14: 99–103. 25 Olive PL, Johnston PJ, Banath JP, Durand RE. The comet assay: a 7 Friesen C, Herr I, Krammer PH, Debatin KM. Involvement of the new method to examine heterogeneity associated with solid CD95 (APO-1/FAS) receptor/ligand system in drug-induced tumors. Nature Med 1998; 4: 103–105. apoptosis in leukemia cells. Nature Med 1996; 2: 574–577. 26 McKelvey-Martin V, Green MH, Schmezer P, Pool ZB, De MM, 8Ro¨thig H-J, Kraemer HP, Sedlacek HH. Aclarubicin: experimental Collins A. The single cell gel electrophoresis assay (comet assay): and clinical experience. Drugs Exp Clin Res 1985; 11: 123–125. a European review. Mutat Res 1993; 288: 47–63. 9 Hilbe W, Thaler J, Eisterer W, Ludescher C, Niederwieser D. Treat- 27 Gille L, Nohl H. Analyses of the molecular mechanism of adria- ment of relapsed and refractory acute myelogenous leukaemia mycin-induced cardiotoxicity. Free Radic Biol Med 1997; 23: with aclacinomycin A (ACA) and etoposide (VP-16). Leukemia Res 775–782. 1994; 18: 655–658. 28 Hershko C, Link G, Tzahor M, Pinson A. The role of iron and iron 10 Harada M, Shibuya T, Teshima T, Murakawa M, Okamura T, Niho chelators in anthracycline cardiotoxicity. Leuk Lymphoma 1993; Y, Gondo H, Hayashi S, Akashi K, Tamura K et al. A randomized 11: 207–214. phase II trial of low-dose aclarubicin vs very low-dose cytosine 29 Monti E, Prosperi E, Supino R, Bottiroli G. Free radical-dependent arabinoside for treatment of myelodysplastic syndromes. Leuke- DNA lesions are involved in the delayed cardiotoxicity induced mia Res 1993; 17: 629–632. by adriamycin in the rat. Anticancer Res 1995; 15: 193–197. 11 Hansen OP, Pedersen BJ, Ellegaard J, Brincker H, Boesen AM, 30 Wolf D, Rotter V. Major deletions in the gene encoding the p53 Christensen BE, Drivsholm A, Hippe E, Jans H, Jensen KB et al. tumor antigen cause lack of p53 expression in HL-60 cells. Proc Aclarubicin plus cytosine arabinoside vs daunorubicin plus cyto- Natl Acad Sci USA 1985; 82: 790–794. sine arabinoside in previously untreated patients with acute 31 Akashi M, Hachiya M, Osawa Y, Spirin K, Suzuki G, Koeffler HP. myeloid leukemia: a Danish national phase III trial. The Danish Irradiation induces WAF1 expression through a p53-independent Society of Hematology Study Group on AML, Denmark. Leukemia pathway in KG-1 cells. J Biol Chem 1995; 270: 19181–19187. 1991; 5: 510–516. 12 Yamada K, Furusawa S, Saito K, Waga K, Koike T, Arimura H, Aoyagi A, Yamato H, Sakuma H, Tsunogake S et al. Concurrent use of granulocyte colony-stimulating factor with low-dose cyto- Reference added in proof sine arabinoside and aclarubicin for previously treated acute myelogenous leukemia: a pilot study. Leukemia 1995; 9: 10–14. 32 McGahon AJ, Costa Pereira AP, Daly L, Cotter TG. Chemothera- 13 Perkins AS, Stern DF. Apoptosis. In: De Vita VT, Hellmann S, peutic drug-induced apoptosis in human leukaemic cells is inde- Rosenberg SA (eds). Cancer: Principles & Practice of Oncology, pendent of the Fas (APO-1/CD95) receptor/ligand system. Br J fifth edn. Lippincott-Raven: Philadelphia, 1997, pp 79–102. Haematol 1998; 101: 539–547.