The efficacy of topoisomerase II-targeted anticancer agents reflects the persistence of drug-induced cleavage complexes in cells.

Type II topoisomerases are enzymes that modulate the topological structure of DNA by generating transient double-stranded breaks in the backbone of the double helix (1-6). In order to maintain genomic integrity during this process, the enzymes form covalent bonds with the 5′-termini of the cleaved DNA (7-9). Compounds that increase the concentration of covalent topoisomerase II-DNA complexes (i.e., cleavage complexes) are known as topoisomerase II poisons (3, 6, 10-13). These agents impact human health in a variety of ways. Some of the most successful anticancer drugs currently in clinical use, including etoposide and doxorubicin, are topoisomerase II poisons (3, 6, 10-13). However, 2–3% of patients treated with regimens that contain these drugs develop specific leukemias that involve translocations at chromosome band 11q23 (11, 13-17). Naturally occurring topoisomerase II-active compounds, such as genistein and other bioflavonoids, are widely consumed constituents of the human diet (18-22). While bioflavonoids are believed to be chemopreventative in adults, epidemiological studies link their consumption during pregnancy to the formation of infant leukemias (23-25). As seen with the anticancer drugs, many of these infant leukemias display 11q23 chromosomal rearrangements (17, 23-25). The anticancer and leukemogenic effects of the above compounds are believed to result from their ability to poison topoisomerase II (6, 13, 17, 24-29). Humans express two isoforms of the enzyme, topoisomerase IIα and β (2-6). Although both appear to be targeted by anticancer and dietary topoisomerase II poisons, the relative contributions of the individual isoforms to cellular outcomes are not well-defined. However, recent studies imply that topoisomerase IIα may play an important role in the induction of cell death by these compounds (30, 31), while topoisomerase IIβ may play a disproportionate role in generating leukemogenic chromosomal translocations (30, 31). Because of their importance to human health, many of the in vitro actions of etoposide and genistein against human topoisomerase IIα and β have been characterized (13, 24, 28, 32-35). In side-by-side comparisons, the two compounds display similar potencies against both enzyme isoforms and induce comparable levels of cleavage complexes in vitro and in cultured human cells (28). Although these findings predict that both agents would have similar physiological effects, the cytotoxic and genotoxic properties of etoposide and genistein have not been compared directly. Therefore, the effects of these drugs on cell viability and the generation of permanent double-stranded DNA breaks were evaluated. Unexpectedly, etoposide was significantly more cytotoxic and genotoxic than genistein in human CEM cells. The differential actions of the two drugs were not related to the effects of genistein on cellular processes outside of its activity against topoisomerase II. Rather, they appear to be due to a longer persistence of cleavage complexes induced by etoposide as compared to genistein. Finally, differences in the persistence of etoposide-induced topoisomerase IIα- and β-DNA cleavage complexes suggest the potential for preferentially targeting the α isoform by employing a schedule that utilizes pulsed drug treatment-recovery cycles.