Mutation Research 738–739 (2012) 45–51 Contents lists available at SciVerse ScienceDirect Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis jo ur n al hom ep a ge: www.elsevier.com/locate/molmut C om mun i ty a ddress: www.elsevier.com/locate/mutres The DNA topoisomerase II catalytic inhibitor merbarone is genotoxic and induces endoreduplication Nuria Pastor a , Inmaculada Domínguez a , Manuel Luís Orta a , Claudia Campanella b , Santiago Mateos a,∗ , Felipe Cortés a a Department of Cell Biology, Faculty of Biology, University of Seville, Avda Reina Mercedes 6, 41012, Spain b Human Anatomy Section, Department of Experimental Medicine, University of Palermo, Italy a r t i c l e i n f o Article history: Received 15 March 2012 Received in revised form 3 July 2012 Accepted 19 July 2012 Available online 15 August 2012 Keywords: Topoisomerase II Catalytic inhibitor Merbarone DNA damage Clastogens Endoreduplication a b s t r a c t In the last years a number of reports have shown that the so-called topoisomerase II (topo II) catalytic inhibitors are able to induce DNA and chromosome damage, an unexpected result taking into account that they do not stabilize topo II-DNA cleavable complexes, a feature of topo II poisons such as etoposide and amsacrine. Merbarone inhibits the catalytic activity of topo II by blocking DNA cleavage by the enzyme. While it was first reported that merbarone does not induce genotoxic effects in mammalian cells, this has been challenged by reports showing that the topo II inhibitor induces efficiently chromosome and DNA damage, and the question as to a possible behavior as a topo II poison has been put forward. Given these contradictory results, and the as yet incomplete knowledge of the molecular mechanism of action of merbarone, in the present study we have tried to further characterize the mechanism of action of merbarone on cell proliferation, cell cycle, as well as chromosome and DNA damage in cultured CHO cells. Merbarone was cytotoxic as well as genotoxic, inhibited topo II catalytic activity, and induced endoreduplication. We have also shown that merbarone-induced DNA damage depends upon ongo- ing DNA synthesis. Supporting this, inhibition of DNA synthesis causes reduction of DNA damage and increased cell survival. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Research has focused on the ubiquitous nuclear enzymes DNA topoisomerases for over 20 years, not only for their reported essen- tial roles in gene regulation, cell cycle, mitosis and chromosome structure, but also for their importance as molecular targets of an increasing number of cancer chemotherapeutic drugs [1]. A num- ber of reviews on the structure and functions of these enzymes in eukaryotic cells have been published [2,3]. As to their functions, DNA topoisomerases are directly involved in basic biological processes depending upon the dynamic nature and topology of the DNA molecule, such as replication, transcrip- tion, recombination, repair and segregation. While topoisomerase I (topo I) relaxes DNA supercoiling by a single-stranded DNA pas- sage mechanism, topo II hydrolyses ATP and changes DNA topology by a double-stranded DNA passage process. This molecular mecha- nism of topo II allows the enzyme not only to resolve supercoiling, but is a feature that makes it unique in its capacity to decatenate ∗ Corresponding authors. Tel.: +34 954554339; fax: +34 954610261. E-mail addresses: smateos@us.es (S. Mateos), cortes@us.es (F. Cortés). intertwined double-stranded DNA molecules and resolve knots that can arise during DNA topological changes. Drugs that affect DNA topoisomerases by interfering with their crucial function on DNA metabolism have proven to be useful for cancer treatment [1,4,5]. Merbarone (5-[N-phenylcarboxamido]- 2-thiobarbituric acid; NSC 336628) has been classified as a DNA topo II catalytic inhibitor that has been proposed to act primar- ily by blocking topo II-mediated DNA cleavage [6]. Accordingly, it was considered as able to prevent cleavage complexes DNA- enzyme from forming (topo II suppressor) and so differs from topo II poisons such as etoposide and amsacrine [1], that stabilize covalent complexes being responsible for topo II-mediated DNA damage, thus converting this essential enzyme into a potent cellu- lar toxin. Supporting this, early reports concluded that merbarone does not induce genotoxic effects in mammalian cells [7–9]. Neverthe- less, this concept has been challenged in the last years on the basis that merbarone has been shown to induce efficiently chro- mosome and DNA damage [10,11], a feature that should not be expected from its reported inhibitory mechanism on DNA cleav- age [6]. Besides, the question as to a possible behavior as a topo II poison has been put forward [11], but as yet the exact molecular mechanism of action of merbarone is debated. 0027-5107/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrfmmm.2012.07.005