XK469 (NSC 697887) is a synthetic quinoxaline phenoxypropionic acid derivative that possesses unusual solid tumor selectivity and activity against multidrug-resistant cancer cells. We report here that XK469 and its S(؊) and R(؉)-isomers induce reversible protein-DNA crosslinks in mammalian cells. Under protein denaturing conditions, the protein-DNA crosslinks are rendered irreversible and stable to DNA banding by CsCl gradient ultracentrifugation. Several lines of evidence indicate that the primary target of XK469 is topoisomerase II␤. Preferential targeting of topoisomerase II␤ may explain the solid tumor selectivity of XK469 and its analogs because solid tumors, unlike leukemias, often have large populations of cells in the G1͞G0 phases of the cell cycle in which topoisomerase II␤ is high whereas topoisomerase II␣, the primary target of many leukemia selective drugs, is low. X K469 (NSC 697887, Fig. 1) is an analog of the herbicide Assure (DuPont) that was discovered in a screen for solid tumor-selective agents (1-3). The screen is based on an agar diffusion assay in which an agent's relative toxicity for leukemias and solid tumors is determined by comparison of zones of colony inhibition (3, 4). A zone difference of 250 units represents approximately an 8-fold difference in sensitivity, and XK469 gave a leukemia͞solid tumor zone difference of 510 (1). XK469 has broad activity against murine solid tumors such as colon 38, pancreatic 03, and mammary 16͞C (1). The R(ϩ)-and S(Ϫ)-isomers of XK469 (respectively, NSC 698215 and NSC 698216) were found to be equally toxic in studies with animal tumor models (1). XK469 also was found to be highly active against multidrug-resistant tumors (1, 2). The dose-limiting toxicities for XK469 and its analogs were marrow toxicity and epithelial damage in the gastrointestinal tract. XK469 has low toxicity in comparison to other anticancer agents, such as camptothecin. For instance, the average in vitro GI 50 (50% growth inhibitory concentration) for the NCI 60 tumor cell line panel is 7 ϫ 10 Ϫ5 M for XK469 and 4.5 ϫ 10 Ϫ8 for camptothecin, a 1,500ϫ difference (National Cancer Institute Developmental Therapeutics Compare web site, http:͞͞dtp.nci.gov). The R-and S-isomers of XK469 were well tolerated by tumor-bearing mice at an effective therapeutic dose of 74.4 mg͞kg, followed by several lower doses of 47 mg͞kg͞injection (1). Thus, relatively high levels of XK469 have been tolerated in animal model studies. Extensive pharmacokinetic studies of the S-isomer of XK469 in CD1F2 mice were performed at 100 mg͞kg (Ϸ1͞6 maximum tolerated total dose) as a single i.v. bolus dose. In these studies, up to 0.48 mg͞ml (Ϸ1.4 mM) maximum serum concentrations of XK469 were produced (K.K.C., unpublished data). Thus, millimolar in vivo concentrations can be produced at nontoxic doses in rodents. The most active analogs, such as XK469, have a halogen at the 7 position (1). The carboxylic acid forms are water soluble and can be injected. Exposure of human colon carcinoma cells to XK469 for 24 hr resul...

I have found that antineoplastic drugs which are known to be inhibitors of mammalian DNA topoisomerases have pronounced and selective effects on simian virus 40 DNA replication. Ellipticine, 4'-(9-acridinylamino)methanesulfon-m-aniside, and Adriamycin blocked decatenation of newly replicated simian virus 40 daughter chromosomes in vivo. The arrested decatenation intermediates produced by these drugs contained single-strand DNA breaks. Ellipticine in particular produced these catenated dimers rapidly and efficiently. Removal of the drug resulted in rapid reversal of the block and completion of decatenation. The demonstration that these drugs interfere with decatenation suggests that they may exert their cytotoxic and antineoplastic effects by preventing the separation of newly replicated cellular chromosomes. Camptothecin rapidly breaks replication forks in growing Cairns structures. It is likely that the target of camptothecin is the "swivel" topoisomerase required for DNA replication and that it is located at or very near the replication fork in vivo. Evidence is presented that many of the broken Cairns structures are in fact half-completed sister chromatid exchanges. One pathway for the resolution of these structures is completion of the sister chromatid exchange to produce a circular head-to-tail dimer.DNA topoisomerases are now recognized as important targets for cancer chemotherapy (for reviews, see references 20 and 32). A number of cytotoxic drugs which were found to have antineoplastic properties have recently been shown to be topoisomerase inhibitors (3,12,15,25,26,31,33). These drugs typically interfere with the breakage-reunion cycles of topoisomerases to produce single-and double-strand DNA breaks. Recent studies have shown a correlation between cytotoxicity and levels of DNA strand breakage caused by these drugs (6,7,21). It is also possible that these drugs can exert their cytotoxic and antineoplastic effects through interference with reactions that require topoisomerases, such as replication fork progression, transcription, and separation (decatenation) of newly replicated daughter chromosomes. The work reported here demonstrates that topoisomerase inhibitors can selectively and reversibly interfere either with replication forks or with the decatenation process in simian virus 40 (SV40) DNA replication. Improved understanding of the modes of action of this class of drugs should aid in the rational design of a second generation of antineoplastic topoisomerase inhibitors.MATERIALS AND METHODS Cell culture and virus infection. African green monkey kidney cells (CV-1) were grown in Eagle minimal essential medium (Gibco) supplemented with 14 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.2) and 4 mM NaHCO3. Cells were infected with SV40 strain 777 at a multiplicity of 10 PFU/cell, and experiments were carried out 36 h after infection at the peak of SV40 DNA replication.Radiolabeling and preparation of viral DNA. Replicating SV40 DNA was pulse-labeled with [methyl-3H]thymidine as in...

An ethanol extract of Psoralea corylifolia caused strong DNA polymerase inhibition in a whole cell bioassay specific for inhibitors of DNA replication enzymes. Bioassay-directed purification of the active compounds led to the isolation of the new compound corylifolin (1) and the known compound bakuchiol (2) as DNA polymerase inhibitors. On the basis of the structures of 1 and 2, resveratrol (3) was tested and found to be active as a DNA polymerase inhibitor in this bioassay. Neobavaisoflavone (4) was isolated as a DNA polymerase inhibitor, daidzein (5) as a DNA polymerase and topoisomerase II inhibitor, and bakuchicin (6) as a topoisomerase II inhibitor.

Antineoplastic bis(dioxopiperazine)s, such as meso-2,3-bis(2,6-dioxopiperazin-4-yl)butane (ICRF-193), are widely believed to be only catalytic inhibitors of topoisomerase II. However, topoisomerase inhibitors have little or no antineoplastic activity unless they are topoisomerase poisons, a special subclass of topoisomerasetargeting drugs that stabilize topoisomerase-DNA strand passing intermediates and thus cause the topoisomerase to become a cytotoxic DNA-damaging agent.