Etoposide metabolites enhance DNA topoisomerase II cleavage near leukemia-associated MLL translocation breakpoints.

Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by epipodophyllotoxins may play a role in the genesis of leukemia-associated MLL gene translocations. We investigated whether etoposide catechol and quinone metabolites can damage the MLL breakpoint cluster region in a DNA topoisomerase II-dependent manner like the parent drug and the nature of the damage. Cleavage of two DNA substrates containing the normal homologues of five MLL intron 6 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIalpha, ATP, and either etoposide, etoposide catechol, or etoposide quinone. Many of the same cleavage sites were induced by etoposide and by its metabolites, but several unique sites were induced by the metabolites. There was a preference for G(-1) among the unique sites, which differs from the parent drug. Cleavage at most sites was greater and more heat-stable in the presence of the metabolites compared to etoposide. The MLL translocation breakpoints contained within the substrates were near strong and/or stable cleavage sites. The metabolites induced more cleavage than etoposide at the same sites within a 40 bp double-stranded oligonucleotide containing two of the translocation breakpoints, confirming the results at a subset of the sites. Cleavage assays using the same oligonucleotide substrate in which guanines at several positions were replaced with N7-deaza guanines indicated that the N7 position of guanine is important in metabolite-induced cleavage, possibly suggesting N7-guanine alkylation by etoposide quinone. Not only etoposide, but also its metabolites, enhance DNA topoisomerase II cleavage near MLL translocation breakpoints in in vitro assays. It is possible that etoposide metabolites may be relevant to translocations.

DNA topoisomerases as targets for the anticancer drug TAS-103: primary cellular target and DNA cleavage enhancement.

TAS-103 is a novel antineoplastic agent that is active against in vivo tumor models [Utsugi, T., et al. (1997) Jpn. J. Cancer Res. 88, 992-1002]. This drug is believed to be a dual topoisomerase I/II-targeted agent, because it enhances both topoisomerase I- and topoisomerase II-mediated DNA cleavage in treated cells. However, the relative importance of these two enzymes for the cytotoxic actions of TAS-103 is not known. Therefore, the primary cellular target of the drug and its mode of action were determined. TAS-103 stimulated DNA cleavage mediated by mammalian topoisomerase I and human topoisomerase IIalpha and beta in vitro. The drug was less active than camptothecin against the type I enzyme but was equipotent to etoposide against topoisomerase IIalpha. A yeast genetic system that allowed manipulation of topoisomerase activity and drug sensitivity was used to determine the contributions of topoisomerase I and II to drug cytotoxicity. Results indicate that topoisomerase II is the primary cellular target of TAS-103. In addition, TAS-103 binds to human topoisomerase IIalpha in the absence of DNA, suggesting that enzyme-drug interactions play a role in formation of the ternary topoisomerase II.drug.DNA complex. TAS-103 induced topoisomerase II-mediated DNA cleavage at sites similar to those observed in the presence of etoposide. Like etoposide, it enhanced cleavage primarily by inhibiting the religation reaction of the enzyme. Based on these findings, it is suggested that TAS-103 be classified as a topoisomerase II-targeted drug.

Binding of etoposide to topoisomerase II in the absence of DNA: decreased affinity as a mechanism of drug resistance.

Despite the prevalence of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, interactions between these agents and topoisomerase II are not well understood. Therefore, to further define interactions between anticancer drugs and the type II enzyme, a nitrocellulose filter assay was used to characterize the binding of etoposide to yeast topoisomerase II. Results indicate that etoposide binds to the enzyme in the absence of DNA. The apparent Kd value for the interaction was approximately 5 microM drug. Etoposide also bound to ytop2H1012Y, a mutant yeast type II enzyme that is approximately 3-4-fold resistant to etoposide. However, the apparent Kd value for the drug (approximately 16 microM) was approximately 3 times higher than that determined for wild-type topoisomerase II. Although it has been widely speculated that resistance to topoisomerase II-targeted anticancer agents results from a decreased drug-enzyme binding affinity, these data provide the first direct evidence in support of this hypothesis. Finally, the ability of yeast topoisomerase II to bind etoposide was dependent on the presence of the hydroxyl moiety of Tyr783, suggesting specific interactions between etoposide and the active site residue that is involved in DNA scission.

In vitro evolution of preferred topoisomerase II DNA cleavage sites.

Topoisomerase II is an essential enzyme that is the target for several clinically important anticancer drugs. Although this enzyme must create transient double-stranded breaks in the genetic material in order to carry out its indispensable DNA strand passage reaction, the factors that underlie its nucleotide cleavage specificity remain an enigma. Therefore, to address the critical issue of enzyme specificity, a modified systematic evolution of ligands by exponential enrichment (SELEX) protocol was employed to select/evolve DNA sequences that were preferentially cleaved by Drosophila melanogaster topoisomerase II. Levels of DNA scission rose substantially (from 3 to 20%) over 20 rounds of SELEX. In vitro selection/evolution converged on an alternating purine/pyrmidine sequence that was highly AT-rich (TATATATACATATATATA). The preference for this sequence was more pronounced for Drosophila topoisomerase II over other species and was increased in the presence of DNA cleavage-enhancing anticancer drugs. Enhanced cleavage appeared to be based on higher rates of DNA scission rather than increased binding affinity or decreased religation rates. The preferred sequence for topoisomerase II-mediated DNA cleavage is dramatically overrepresented ( approximately 10,000-fold) in the euchromatic genome of D. melanogaster, implying that it may be a site for the physiological action of this enzyme.

Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme.

Topoisomerase II is a ubiquitous enzyme that is essential for the survival of all eukaryotic organisms and plays critical roles in virtually every aspect of DNA metabolism. The enzyme unknots and untangles DNA by passing an intact helix through a transient double-stranded break that it generates in a separate helix. Beyond its physiological functions, topoisomerase II is the target for some of the most active and widely prescribed anticancer drugs currently utilized for the treatment of human cancers. These drugs act in an insidious fashion and kill cells by increasing levels of covalent topoisomerase II-cleaved DNA complexes that are normally fleeting intermediates in the catalytic cycle of the enzyme. Over the past several years, we have made considerable strides in our understanding of the catalytic mechanism of topoisomerase II and the mechanism of action of drugs targeted to this enzyme. These advances have provided novel insights into the physiological functions of topoisomerase II and have led to the development of more efficacious chemotherapeutic regimens and novel anticancer drugs. Considering the importance of topoisomerase II to the eukaryotic cell and to cancer chemotherapy, it is essential to understand its enzymatic function and pharmacological properties. Therefore, this review will discuss the mechanism of action of eukaryotic topoisomerase II and topoisomerase II-targeted drugs.

Quinolones share a common interaction domain on topoisomerase II with other DNA cleavage-enhancing antineoplastic drugs.

Topoisomerase II is the cytotoxic target for a number of clinically relevant antineoplastic drugs. Despite the fact that these agents differ significantly in structure, a previous study [Corbett, A. H., Hong, D., & Osheroff, N. (1993) J. Biol. Chem. 268, 14394-14398] indicated that the site of action for etoposide on topoisomerase II overlaps those of other DNA cleavage-enhancing drugs. Therefore, to further define interactions between drugs and the enzyme, the functional interaction domain (i.e., interaction domain defined by drug function) for quinolones on Drosophila topoisomerase II was mapped with respect to several classes of antineoplastic agents. This was accomplished by characterizing the effects of ciprofloxacin (a gyrase-targeted antibacterial quinolone) on the ability of etoposide, amsacrine, genistein, and the antineoplastic quinolone, CP-115,953, to enhance topoisomerase II-mediated DNA cleavage. Although ciprofloxacin interacts with the eukaryotic type II enzyme, it shows little ability to stimulate DNA cleavage. Ciprofloxacin attenuated cleavage enhancement by all of the above drugs. Similar results were obtained using a related quinolone, CP-80,080, as a competitor. In addition, kinetic analysis of DNA cleavage indicated that ciprofloxacin is a competitive inhibitor of CP-115,953 and etoposide. Finally, ciprofloxacin inhibited the cytotoxic actions of CP-115,953 and etoposide in mammalian cells to an extent that paralleled its in vitro attenuation of cleavage. These results strongly suggest that several structurally disparate DNA cleavage-enhancing antineoplastic drugs share an overlapping site of action on topoisomerase II. Based on the results of drug competition and mutagenesis studies, a model for the drug interaction domain on topoisomerase II is described.

Topoisomerase II.etoposide interactions direct the formation of drug-induced enzyme-DNA cleavage complexes.

Topoisomerase II is the target for several highly active anticancer drugs that induce cell death by enhancing enzyme-mediated DNA scission. Although these agents dramatically increase levels of nucleic acid cleavage in a site-specific fashion, little is understood regarding the mechanism by which they alter the DNA site selectivity of topoisomerase II. Therefore, a series of kinetic and binding experiments were carried out to determine the mechanistic basis by which the anticancer drug, etoposide, enhances cleavage complex formation at 22 specific nucleic acid sequences. In general, maximal levels of DNA scission (i.e. Cmax) varied over a considerably larger range than did the apparent affinity of etoposide (i.e. Km) for these sites, and there was no correlation between these two kinetic parameters. Furthermore, enzyme.drug binding and order of addition experiments indicated that etoposide and topoisomerase II form a kinetically competent complex in the absence of DNA. These findings suggest that etoposide. topoisomerase II (rather than etoposide.DNA) interactions mediate cleavage complex formation. Finally, rates of religation at specific sites correlated inversely with Cmax values, indicating that maximal levels of etoposide-induced scission reflect the ability of the drug to inhibit religation at specific sequences rather than the affinity of the drug for site-specific enzyme-DNA complexes.

Increased drug affinity as the mechanistic basis for drug hypersensitivity of a mutant type II topoisomerase.

Altered sensitivity of topoisomerase II to anticancer drugs profoundly affects the response of eukaryotic cells to these agents. Therefore, several approaches were employed to elucidate the mechanism of drug hypersensitivity of the mutant yeast type II topoisomerase, top2H1012Y. This mutant, which is approximately 5-fold hypersensitive to ellipticine, formed DNA cleavage complexes more rapidly than the wild-type yeast enzyme in the presence of the drug. Conversely, no change in the rate of DNA religation was observed. There was, however, a correlation between increased cleavage rates and enhanced drug binding affinity. The apparent dissociation constant for ellipticine in the mutant topoisomerase II.drug.DNA ternary complex was approximately 5-fold lower than in the wild-type ternary complex. Furthermore, the apparent KD value for the mutant binary (topoisomerase II.drug) complex was approximately 2-fold lower than the corresponding wild-type complex, indicating that drug hypersensitivity is intrinsic to the enzyme. These findings strongly suggest that the enhanced ellipticine binding affinity for topoisomerase II is the mechanistic basis for drug hypersensitivity of top2H1012Y.

Phosphorylation of the alpha- and beta-isoforms of DNA topoisomerase II is qualitatively different in interphase and mitosis in Chinese hamster ovary cells.

Qualitative differences between interphase and mitotic topoisomerase II were studied in Chinese hamster ovary cells. Differences in sites of phosphorylation of in vivo 32P-labeled topoisomerase II alpha were observed between mitosis and interphase by one-dimensional phosphopeptide mapping of partial tryptic digests. Two-dimensional phosphopeptide mapping of complete trypsin digests revealed two phosphopeptides unique to interphase and three phosphopeptides unique to mitosis. A reduced electrophoretic mobility on denaturing gels (approximately 190 kDa) was observed for the beta-isoform of topoisomerase II in mitosis relative to interphase. Treatment of lysates with alkaline phosphatase demonstrated that this was due to phosphorylation of mitotic topoisomerase II beta. The existence of interphase- and mitosis-specific sites of phosphorylation of topoisomerase II alpha, along with the electrophoretic mobility shift caused by phosphorylation of topoisomerase II beta in mitosis, demonstrates qualitative differences between interphase and mitosis in the phosphorylation state of both isoforms of topoisomerase II.

Cell-cycle-dependent phosphorylation and activity of Chinese-hamster ovary topoisomerase II.

Cell-cycle-dependent protein levels and phosphorylation of DNA topoisomerase II in relation to its catalytic and cleavage activities were studied in Chinese-hamster ovary cells. Immunoreactive topoisomerase II protein levels were maximal in G2-phase cells, intermediate in S- and M-phase cells, and minimal in a predominantly G1-phase population. When the phosphorylation of topoisomerase II in vivo was corrected for differences in specific radioactivity of intracellular ATP, the apparent phosphorylation of S- and M-phase topoisomerase II was altered significantly. Relative phosphorylation in vivo was found to be greatest in M-phase cells and decreased in the other populations in the order: S > G2 > asynchronous. Phosphoserine was detected in every phase of the cell cycle, with a minor contribution of phosphothreonine demonstrated in M-phase cells. Topoisomerase II activity measured in vivo as 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-demethylepipodophylloto xin (VP-16)-induced DNA double-strand breaks (determined by neutral filter elution) increased in the order: asynchronous < S < G2 < M. Topoisomerase II cleavage activity, assayed in vitro as the formation of covalent enzyme-DNA complexes, was lowest in S phase, intermediate in asynchronous and G2-phase cells, and maximal in M phase. Topoisomerase II decatenation activity was 1.6-1.8-fold greater in S-, G2- and M-phase populations relative to asynchronous cells. Therefore DNA topoisomerase II activity measured both in vivo and in vitro is maximal in M phase, that phase of the cell cycle with an intermediate level of immunoreactive topoisomerase II but the highest level of enzyme phosphorylation. The discordance between immunoreactive topoisomerase II protein levels, adjusted relative phosphorylation, catalytic activity, cleavage activity and amino acid residue(s) modified, suggests that the site of phosphorylation may be cell-cycle-dependent and critical in determining catalytic and cleavage activity.