Induction of Genotoxic and Cytotoxic Damage by Aclarubicin, a Dual Topoisomerase Inhibitor N

Mutation Research 583 (2005) 26–35 Induction of genotoxic and cytotoxic damage by aclarubicin, a dual topoisomerase inhibitor N. Hajji, S. Mateos, N. Pastor, I. Dom´ınguez, F. Cortes´ ∗ Department of Cell Biology, Faculty of Biology, University of Seville, Avda. Reina Mercedes No. 6, 41012 Seville, Spain Received 19 May 2004; received in revised form 11 September 2004; accepted 21 January 2005 Available online 11 March 2005 Abstract The anthracycline aclarubicin (ACLA) is an intercalative antibiotic and antineoplastic agent that efficiently binds to DNA, leading to a secondary inhibition of the catalytic activity of topoisomerase II (topo II) on DNA. Besides this activity, ACLA has been reported to exert a concomitant poisoning effect on topo I, in a fashion similar to that of the antitumor drug camptothecin and its derivatives. As a consequence of this dual (topo II catalytic inhibiting/topo I poisoning) activity of ACLA, the picture is somewhat confusing with regards to DNA damage and cytotoxicity. We studied the capacity of ACLA to induce catalytic inhibition of topo II as well as cytotoxic effects and DNA damage in cultured Chinese hamster V79 cells and their radiosensitive counterparts irs-2. The ultimate purpose was to find out whether differences could be observed between the two cell lines in their response to ACLA, as has been widely reported for radiosensitive cells treated with topo poisons. Our results seem to agree with the view that the radiosensitive irs-2 cells appear as hypersensitive ACLA as compared with radiation repair-proficient V79 cells. The recovery after ACLA treatment was also followed-up, and the irs-2 mutant was found to be less proficient than V79 to repair DNA strand breaks induced by ACLA. © 2005 Elsevier B.V. All rights reserved. Keywords: Aclarubicin; Topoisomerase II; Topoisomerase I; Cytotoxicity; DNA damage 1. Introduction II catalytic inhibitors. While topo II poisons are well known for their ability to efficiently induce DNA strand Topoisomerase II (topo II)-targeting drugs are clas- breaks [1], catalytic inhibitors generally act at stages sified into either topo II poisons, which play a role in in the catalytic cycle of the enzyme where both DNA stabilizing the otherwise fleeting enzyme-DNA inter- strands are intact and, therefore by definition they are mediates, the so-called ‘cleavable complexes’, or topo not expected to cause any DNA breakage [2]. While topo II poisons have been thoroughly studied for their mechanism(s) of interference with topo II – some of the ∗ Corresponding author. Tel.: +34 95 4557039; fax: +34 95 4610261. more potent and widely used anticancer drugs belong E-mail address: [email protected] (F. Cortes).´ to this category – much less is known about catalytic 1383-5718/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2005.01.012 N. Hajji et al. / Mutation Research 583 (2005) 26–35 27 inhibitors and their relationship with topo II, or with re- ACLA in two cultured Chinese hamster lung fibroblast spect to their clinical use [3]. Nonetheless, aclarubicin cell lines, namely the parental line V79, which repairs (ACLA) represents an exception in that it is a topo II DNA radiation damage normally, and its radiosensitive catalytic inhibitor that has been successfully used in mutant irs-2 [21], to see whether the effects of ACLA clinical oncology practice. This intercalative antibiotic are different between these two cell lines. belongs to the class of anthracyclines, and efficiently binds to DNA, which in turn results in an inhibition of topo II to access the DNA [4–6], i.e. ACLA is a DNA 2. Materials and methods intercalator that indirectly interferes with topo II. On the other hand, besides its role as a topo II catalytic in- 2.1. Cells and culture conditions hibitor, an interesting feature of ACLA is its reported concomitant action as a topo I poison through stabi- The parental lung fibroblast Chinese hamster cell lization of the topo I-DNA cleavable complex [7–10]. line V79 was purchased from the American Type This dual mechanism of action of this antibiotic, Culture Collection (ATCC), USA. The radiosensi- involving both topo II (catalytic inhibition) and topo tive mutant irs-2 was kindly provided by Dr. John I (poisoning) makes this drug an interesting choice Thacker (Medical Research Council, Harwell, UK). for the assessment of the relative importance of ei- Cells were routinely maintained as monolayer in Min- ther enzyme in DNA function and of the deleteri- imum Essential Medium (Gibco BRL) supplemented ous consequences of topoisomerase dysfunction as a with 10% fetal bovine serum, 2 mM l-glutamine, and whole. Besides, the dual topo I poisoning-topo II cat- the antibiotics penicillin (50 units/ml) and strepto- alytic inhibition exerted by ACLA contrasts with that mycin (50 ␮g/ml). Cells were cultured in a dark en- of other agents acting in a dual fashion, with either vironment at 37 ◦C in an atmosphere containing 5% a poison–poison [7,11–13] or catalytic–catalytic [10] CO2. On regular testing, cell cultures were found to be anti-DNA topoisomerase mechanism. free from mycoplasma. The early notion that catalytic inhibitors of topo II do not induce DNA breaks through stabilization of the 2.2. Cell viability cleavable complex [2,14], because they interfere with the cycle of the enzyme when both DNA strands are 2.2.1. Sulforhodamine B (SRB) assay intact, has been challenged recently. Reports on e.g., V79 and irs-2 cells in the exponential growth phase the bis-dioxopiperazine ICRF-193 described the in- were harvested using trypsin-EDTA (Gibco BRL), duction of chromosomal aberrations [15,16] as well and resuspended in medium. They were seeded at as DNA strand breaks [17–19], raising questions as to 5 × 103 cells/100 ␮l in 96-well microtitre plates (Nunc) the existence of ‘clean’ catalytic inhibitors of topo II and allowed 24 h to attach. Then they were incubated that act on the enzyme without concomitantly inducing further for 48 h in the presence of the DNA topo II in- DNA damage, either directly or indirectly. Focusing on hibitor ACLA. The concentration range tested (0.006 to ACLA, even though studies on this intercalative antibi- 0.37 ␮M) was prepared in tissue culture medium from otic are scarce, its ability to induce DNA damage in two a 1 mM ACLA stock solution. human myeloid cell lines has been measured by use of Following the recommendations of the National the comet assay and reported to correlate with the in- Cancer Institute (USA), the analysis of cytotoxic ef- duction of apoptosis [6]. fects induced by ACLA was conducted by use of a cell The sensitivity of radiosensitive mammalian cell growth test, the SRB assay, as described previously lines to topoisomerase poisons was shown to be sim- [24,25]. Briefly, 50 ␮l/well of cold 50% trichloroacetic ilar to that observed in response to radiation damage acid (TCA) (final concentration 10%) was added to [20–23]. In the case of ACLA, however, taking into ac- the culture and incubated at 4 ◦C for 1 h, to precipi- count the dual nature of this drug as topo II catalytic tate proteins and to fix the cells. The supernatant was inhibitor as well as topo I poison, the picture that arises then discarded, and the plates were washed five times is somewhat confusing. In the present report, we have with deionized water and air-dried. The cells were then evaluated the cytotoxic and/or genotoxic activity of stained with 100 ␮l/well of 0.4% SRB dissolved in 1% 28 N. Hajji et al. / Mutation Research 583 (2005) 26–35 acetic acid for 30 min at room temperature. Unbound were obtained by centrifugation at 2000 rpm (Eppen- SRB was removed by washing five times with 1% acetic dorf centrifuge), for 5 min at 4 ◦C. Nuclei were then acid, and then the plates were air-dried. The stained washed in 1 ml of nucleus wash buffer (5 mM potas- protein was solubilized in 100 ␮l/well of 10 mM un- sium phosphate buffer, pH 7.5, 1 mM phenylmethyl buffered Tris base by shaking. The optical density was sulfonyl fluoride (PMSF), 1 mM ␤-mercaptoethanol, read at 492 nm using a microtitre plate reader (ELISA). and 0.5 mM dithiothreitol (DTT)). The nuclei were then Similarly, cells from both lines were treated with dif- pelleted as described above and resuspended in 50 ␮l ferent doses (0.005–2.5 ␮M) of the bisdioxopiperazine of nucleus wash buffer, and 50 ␮l of 4 mM EDTA was ICRF-193 in order to assess the relative importance for added. Following incubation at 0 ◦C for 15 min, the nu- cytotoxicity of inhibition of topo II catalytic activity clei were lysed by adding 100 ␮l of 2 M NaCl, 20 mM compared with poisoning of topo I. Each type of ex- Tris–HCl pH 7.5, 10 mM ␤-mercaptoethanol, 1 mM periment was independently performed in triplicate. PMSF. Following a 15 min incubation at 0 ◦C, 50 ␮l of 18% polyethylene glycol (PEG-6000) in 1 M NaCl, 2.2.2. Clonogenic assay 50 mM Tris–HCl pH 7.5, 10 mM ␤-mercaptoethanol, V79 and irs-2 cells in the exponential growth phase and 1 mM PMSF were added. The suspension was in- were trypsinized at 37 ◦C for 5 min, and pipetted five cubated for a further 40 min period at 0 ◦C. Then the times to remove cell clumps in order to obtain a single- supernatant from a 30 min centrifugation at 12,500 rpm cell suspension. The cells were then counted and seeded at 4 ◦C was collected.

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