Topoisomerase I (Top1) is an abundant and essential enzyme. Top1 is the selective target of camptothecins, which are effective anticancer agents. Top1-DNA cleavage complexes can also be trapped by various endogenous and exogenous DNA lesions including mismatches, abasic sites and carcinogenic adducts. Tyrosyl-DNA phosphodiesterase (Tdp1) is one of the repair enzymes for Top1-DNA covalent complexes. Tdp1 forms a multiprotein complex that includes poly(ADP) ribose polymerase (PARP). PARP-deficient cells are hypersensitive to camptothecins and functionally deficient for Tdp1. We will review recent developments in several pathways involved in the repair of Top1 cleavage complexes and the role of Chk1 and Chk2 checkpoint kinases in the cellular responses to Top1 inhibitors. The genes conferring camptothecin hypersensitivity are compiled for humans, budding yeast and fission yeast. A. Introduction: Mammalian Topoisomerase Families, Top1 Functions and Catalytic MechanismsSeven topoisomerase genes are encoded in the human nuclear genome [1]. The enzymes (abbreviated Topo or Top) have been numbered in the order of their discovery except for the most recent enzyme, mitochondrial topoisomerase I (Top1mt) [2,3]. Vertebrate cells contain two Top1 (Top1 for the nuclear genome and Top1mt for the mitochondrial genome), two Top2 (Top2α and β) and two Top3 (Top3α and β). The seventh topoisomerase is Spo11, whose expression is restricted to germ cells. Top3α forms heterodimers with BLM (the gene product deficient in Bloom syndrome) and is functionally related to the resolution of post-replicative hemicatenanes and recombination intermediates [4,5]. Top1 proteins belong to the family of the tyrosine recombinases (which includes λ-integrase, Flip and Cre recombinases), and Top2 is related to bacterial gyrase and Topo IV, which are the targets of quinolone antibiotics.Topoisomerases and tyrosine recombinases nick and religate DNA by forming a covalent enzyme-DNA intermediate between an enzyme catalytic tyrosine residue and the end of the broken DNA (Fig. 1). These covalent intermediates are generally referred to as "cleavage (or cleavable) complexes" (Fig. 2). Topoisomerases have also been classified in two groups depending whether they cleave and religate one strand (type I) or both strands (type II) of the DNA duplex. Type I enzymes include Top1 (nuclear), Top1mt, Top3α and β and type II enzymes include Top2α and β and Spo11.Top1 is essential in vertebrates and flies but not in yeast. Knocking out the TOP1 gene results in early embryonic lethality in mouse [6] and fly [7]. By contrast, yeast survives in the absence *To whom reprint requests should be addressed, Bldg. 37, Rm. 5068, NIH, Bethesda, MD 20892-4255 [8]. Top1 is expressed constitutively throughout the cell cycle [9] and is concentrated in the nucleolus [10,11]. Its main function is to relieve both positive and negative DNA supercoiling generated by transcription and replication, and possibly DNA repair and chromatin remodeling [1,[12][13][14]. The mechanistic sim...

Camptothecin (CPT) derivatives are effective anticancer drugs, especially against solid tumors. As CPTs are chemically unstable and have clinical limitations, we have synthesized indenoisoquinolines as novel topoisomerase I (Top1) inhibitors. We presently report two indenoisoquinoline derivatives, NSC 725776 and NSC 724998, which have been selected for therapeutic development. Both are potent Top1 inhibitors and induce Top1 cleavage at unique genomic positions compared with CPT. Consistent with Top1 poisoning, protein-linked DNA breaks were detected in cells treated with NSC 725776 and NSC 724998 at nanomolar concentrations. Those druginduced protein-linked DNA breaks persisted longer after drug removal than those produced by CPT. Studies in human cells in culture show that NSC 725776 and NSC 724998 exert antiproliferative activity at submicromolar concentrations. Furthermore, NSC 725776 and NSC 724998 show crossresistance in cells deficient or silenced for Top1, which is consistent with their selective Top1 targeting. Similar to other known Top1 inhibitors, NSC 725776-treated and NSC 724998-treated cells show an arrest of cell cycle progression in both S and G 2 -M and a dependence on functional p53 for their cytotoxicity. Dose-dependent ;-H2AX foci formation was readily observed in cells treated with NSC 725776 and NSC 724998. These ;-H2AX foci were detectable at pharmacologically relevant doses for up to 24 h and thus could be used as biomarkers for clinical trials (phase 0). [Cancer Res 2007; 67(21):10397-405]