Eighth conference on DNA topoisomerases and therapy Amsterdam, The Netherlands 15–17 October 1997

Abstract

During the last 4 years this conference has been held annually, alternating between the New York University in NewYork, USA and the University HospitalVrije Universiteit in Amsterdam, The Netherlands.The change from a biannual to an annual conference was necessary to accommodate a logarithmically increasing fundamental knowledge on topoisomerases and has followed the successfill introduction of several novel topoisomerase inhibitors in the clinic. Accordingly, the conference attempted to combine preclinical with clinical research, with the purpose to promote discussion between research scientists and physicians. Here we summarize some of the conference highlights. Topoisomerase structures and functions. Progress in this area is increasingly being used in the development of new topoisomerase inhibitors.The role of topoisomerase (topo) II and IV in DNA replication was discussed by N. Cozzarelli (University of California, Berkley, CA, USA). Knock-out mice for the topo II~ and for topo IIIc~ genes, following the discovery of two isoforms of this new DNA topoisomerase in the human genome, were reported byJ.Wang (Harvard University, Cambridge, MA, USA); for both genes the homozygous forms are non-viable.The heterozygous topo II~ knock-out mice appear normal.The only alteration is at the skeletal muscle level, probably linked to a nerve defect rather than to muscle damage, implying a possible function of topo II~ in neuronal development for which topo II(z can not substitute. Topo III~, like topo IIc~, is essential and the heterozygous knock-out mice die before birth. U. Laemmli (University of Geneva, Geneva, Switzerland) reported recent fmdings concerning the involvement of DNA topoisomerases in the maintenance of the structure of chromosome loops. Other reports covered the interaction of topoisomerases with other proteins in the correct segregation of chromosomes at mitosis, such as the Barren protein (H.J. Bellen, Howard Hughes Medical Institute, Houston, TX, USA), and in chromosome condensation, such as the smc (structural maintenance of chromosomes) family of proteins (T.Hirano, Cold Spring Harbor, N~, USA).A new enzyme, named topoisomerase VI, was discovered in archeabacteria and its eukaryotic homologs were described (12. Forterre, Universit~ Paris-Sud, Orsay, France). A. Maxwell (University of Leicester, Leicester, UK) discussed the crystal structure of a 59 kDa protein fragment of Escherichia coil DNA gyrase at 2.8 ~k resolution.This bacterial type II topoisomerase is the target of potent antibacterial drugs such as the quinolones.The presented structure includes the enzyme active site and the putative quinolone-binding site. Comparison to a previously published structure of the yeast topoisomerase II revealed a distinct quaternary architecture, and two main conformations of the enzyme catalytic cycle were proposed for the two topoisomerases.Amino acid mutations in the quinolone binding site clustered at a new protein dimer interface, providing insights to how these agents may interfere with the function of the enzyme-DNA complex. Studies of the role of the helicase Sgs 1 and topoisomerases in maintaining genome stability were reported by R. Rothstein (Columbia University, NY,, USA) and I. Hickson (ICRF Laboratories, Oxford, UK), including their implication in the molecular pathogenesis of Bloom's and Wemer's syndromes in humans. Topoisomerase poisons and inhibi tors . Topoisomerase-int eracting drugs are divided into two classes, based on their mechanism of action: poisons and catalytic inhibitors.The former stabilize the cleavable DNA-enzyme complex and stimulate DNA cleavage while the latter inhibit the catalytic reaction without stimulating DNA cleavage. Many effective antitumor and antibacterial drugs are topoisomerase poisons.A major area of the conference was the design and development of novel topoisomerase inhibitors with activity in human cancers and infectious diseases. Sites of DNA cleavage stimulated by structurally-unrelated poisons are highly drug-specific. Drug requirement for specific nucleotides adjacent to the strand cut is essential and specific for drug cleavage stimulation.A model was postulated by G. Capranico (Istituto Nazionale Tumori, Milan, Italy) in which poisons form ternary complexes with DNA and the enzyme active sites, in close contact with DNA base pairs adjacent to the cleaved bond.These in vitro studies were confirmed by in vivo experiments. N. Osheroff (Vanderbilt University, Nashville,TN, USA) reviewed studies on the major forms of spontaneous DNA damage, induced by apurinic and apyrimidinic sites, deaminated cytosines.These alterations stimulated double-stranded DNA cleavage up to 20-fold without altering the sequence specificity of topoisomerase II. DNA damage must therefore be located within the 4-base overhang generated by the enzyme-mediated scission. Apurinic sites were even more effective than etoposide in stimulating in vitro DNA cleavage at sites proximal to human chromosomal 11 q23 leukemic breakpoint.This finding suggests that spontaneous DNA lesions may be able to trigger not only secondary leukemias but also primary leukemias. A special session was dedicated to new topoisomerase inhibitors.Two approaches were used to discover new compounds: (a) a yeast-based assay system for screening natural or synthetic compotmds (B. van Hille et al, Pierre Fabre, Castres, France); (b) rational design of novel analogs of known agents such as acridines