Mechanism of Action of Topoisomerase II-Targeted Antineoplastic Drugs

Mechanism of Action of Topoisomerase IITargeted A n tineoplastic Drugs Neil Osheroff, Anita H. Corbett,' and Megan J. Robinson2 Department of Biochemistry Vanderbilt University School of Medicine Nashville, Tennessee 37332-0146

1. Introduction The topological state of DNA in all living systems is modulated by highly conserved enzymes known as topoisomerases (Wang, 1985; Osheroff, 1989a; Sutcliffe, 1989; Osheroff et al., 1991; Reece and Maxwell, 1991). The type I1 enzyme is required for proper chromosome structure and segregation, is involved in most processes of DNA metabolism, and is essential for the survival of eukaryotic cells (DiNardo et al., 1984; Goto and Wang, 1984; Berrios et al., 1985; Earnshaw and Heck, 1985; Earnshaw et al., 1985; Holm et al., 1985; Wang, 1985, 1991; Gasser and Laemmli, 1986; Gasser et al., 1986; Uemura and Yanagida, 1984, 1986; Uemura et al., 1987; Bae et al., 1988; Dillehay et al., 1989; Rose et al., 1990; Wang et al., 1990). However, the importance of topoisomerase I1 extends beyond its critical physiological functions. Indeed, the enzyme is the primary cellular target for a number of clinically relevant antineoplastic agents, many of which are highly active against human cancers (Liu, 1989; Schneider et al., 1990). I Present address: Department of Cellular and Molecular Biology, Dana Farber Cancer Institute, Harvard University School of Medicine, Boston, Massachusetts 021 15. Present address: Department of Biochemistry, University of Texas, Southwestern Medical Center, Dallas, Texas 75235.

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Advances in Pharmacology, Volume 298 Copyright 0 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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II. Topoisomerase Il-Targeted Antineoplastic Drugs Topoisomerase 11-targeted drugs fall into several structurally diverse classes. A few representative compounds are shown in Fig. 1. Among the antineoplastic drugs that target the type I1 enzyme are the intercalative agents amsacrine, mitoxantrone, and Adriamycin (Adria Laboratories Columbus, OH), which are representative of the anilinoacridines, anthracenediones, and anthracyclines, respectively (Waring, 1981; Wilson et al., 1981; Nelson et al., 1984; Tewey et al., 1984a), and the nonintercalative agents genistein, etoposide, and CP-115,953, which are representative of the isoflavones, demethylepipodophyllotoxins, and quinolones, respectively (Chen et al., 1984; Ross et al., 1984; Chow et al., 1988; Yamashita et al., 1990; Robinson et al., 1991). Classes of topoisomerase 11-targeted drugs not shown in Fig. 1 include the ellipticines, actinomycins, and benzisoquinolinediones, all of which are intercalative, and the nitroimidazoles, which are nonintercalative (Waring, 1981; Tewey et al., 1984b; Hsiang et al., 1989; Liu, 1989; Schneider ef al., 1990; Slrensen er al., 1990). Although many of the above compounds inhibit the overall catalytic activity of topoisomerase I1 (Chen et al., 1984; Nelson et al., 1984; Tewey er

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Fig. 1 Structures of some representative topoisomerase 11-targeted antineoplastic drugs.

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al., 1984a,b; Pommier et al., 1985b; Markovits et al., 1989; Robinson et al., 1991),their cytotoxic potentials do not result from a simple elimination of enzyme activity. All of the evidence currently available points to a far more insidious mode of action; namely, these compounds appear to kill cells by converting topoisomerase I1 into a cellular poison (Liu, 1989; Schneider et al., 1990; Corbett et a!., 1993). As detailed below, this is accomplished by stabilizing covalent enzyme-cleaved DNA complexes that are normal intermediates in the catalytic cycle of topoisomerase I1 (Liu, 1989; Osheroff, 1989a; Gale and Osheroff, 1990; Schneider et al., 1990; Corbett et al., 1993; Andersen et al., 1991; Osheroff et al., 1991). The hypothesis that drugs act by corrupting rather than by eliminating the activity of topoisomerase I1 was first proposed by Kreuzer and Cozzarelli (1979) and can be traced to the pioneering studies on the effects of nalidixic acid (a first-generation quinolone) on DNA gyrase (Gellert et al., 1977; Sugino et al., 1977). Due to the mechanism of drug action, cells that are treated with topoisomerase II-targeted agents accumulate high levels of protein-associated breaks in their genetic material (Liu, 1989; Schneider et al., 1990; Corbett et al., 1993). Thus, the higher the physiological content of the type I1 enzyme, the more potent the effect of drugs (Bodley et al., 1987; Sullivan et al., 1987; Davies et al., 1988; Potmesil et al., 1988; Deffie et al., 1989; Friche et al., 1991; Fry et al., 1991; Webb et al., 1991; Elsea et al., 1992). Since rapidly proliferating or neoplastic cells usually contain elevated enzyme levels (Duget et al., 1983; Heck and Earnshaw, 1986; Bodley et al., 1987; Nelson et a!., 1987; Sullivan et al., 1987; Hsiang et al., 1988; Holden et al., 1990), clinically aggressive tumors appear to be most sensitive to these agents. Despite the importance of topoisomerase II-targeted drugs to the treatment of human cancers, relatively little is known concerning the mechanism by which these structurally disparate compounds alter the catalytic properties of the enzyme. Clearly, before the clinical potential of topoisomerase II-targeted agents can be fully exploited, the mechanism by which these drugs exert their effects must be understood. As a prelude to addressing drug action, however, it is necessary to appreciate how the enzyme target of these compounds carries out its catalytic function. Therefore, the following section will acquaint the reader with the catalytic cycle of topoisomerase 11.

111. Catalytic Cycle of Topoisomerase II Topoisomerase I1 interconverts topological states of DNA by passing an intact double helix through a transient break that it generates in a separate

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helix (Wang, 1985; Osheroff, 1989a; Osheroff et al., 1991). As a consequence of its double-stranded DNA passage reaction, the type I1 enzyme can remove superhelical twists (negative or positive) from DNA as well as resolve inter- or intramolecular nucleic acid tangles. The double-stranded DNA passage reaction takes place at the expense of ATP hydrolysis and requires a divalent cation (Hsieh and Brutlag, 1980: Osheroff rf af.,1983; Osheroff, 1987). Although the catalytic mechanism of the enzyme appears to be concerted and quite complex, it can be dissected into at least six discrete and straightforward steps (Osheroff, 1989a;Osheroff rt al., 1991). The reaction steps that comprise the catalytic cycle of topoisomerase 11 are shown in Fig. 2. A brief description of each follows.

A. Step 1: DNA Binding Topoisomerase I1 recognizes its nucleic acid substrate and in the absence of any cofactors binds to DNA at points of helix-helix juxtaposition (Osheroff and Brutlag, 1983;Osheroff, 1986,1987; Zechiedrich and Osheroff, 1990; Howard et uf., 1991; Roca et af., 1993). Presumably, one of the helices in the enzyme-DNA complex is destined to be cleaved by

'ATP

Fig. 2 Catalytic cycle of topoisomerase I I (Osheroff et d., 1991).The homodimeric enzyme is represented by the croissant-shaped structure. The change in enzyme structure that takes place following step 3 represents the structural transition that occurs upon ATP binding (Lindsley and Wang, 1991). The double-stranded DNA passage reaction of topoisomerase I1 is made up of at least six steph: (1) substrate recognition and binding; (2) pre-strand passage DNA cleavageireligation: (3) double-stranded DNA passage: (4) post-strand passage DNA cleavageheligation; (5) ATP hydrolysis; and (6) enzyme turnover. Transient enzyme-DNA cleavage complexes are shown in brackets.

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topoisomerase 11, while the other is destined to be passed through the break. The enzyme interacts with the cleavage helix at preferred sites (Sander and Hsieh, 1983, 1985; Muller et al., 1988; Andersen et al., 1989; Lee et al., 1989; Capranico e f id., 1990; Thomsen et al., 1990), but the DNA structures that provide specificity have yet to be determined. It is not known whether topoisomerase IT recognizes any specific feature of the passage helix.

B. Step 2: Pre-Strand Passage DNA Cleavage/Religation In the presence of a divalent cation (magnesium is used in uiuo), topoisomerase I1 introduces a transient double-stranded break in the cleavage helix (Liu e f al., 1983; Sander and Hsieh, 1983; Osheroff, 1987). Although at this point in the cycle the type IT enzyme is not competent to carry out DNA strand passage, the presence of the passage helix is a prerequisite for efficient nucleic acid cleavage (Corbett et al., 1992). Sites of enzymemediated DNA breakage correspond to sites of binding on the cleavage helix (Lee et al., 1989; Thomsen el ul., 1990), and double-stranded cleavage leaves a 4-base 5 ’ stagger on the cut DNA (Liu et a/., 1983; Sander and Hsieh, 1983). Several lines of evidence indicate that double-stranded DNA breaks result from the production of two coordinated and sequential nicks made in the cleavage helix (Muller et al., 1988; Andersen et al., 1989; Lee et al., 1989; Zechiedrich rt al., 1989). The DNA cleavage/ religation equilibrium that is established can be readily reversed by the addition of salt, the removal of magnesium, or the shift to suboptimal reaction temperatures (Liu et al., 1983; Sander and Hsieh, 1983; Osheroff and Zechiedrich, 1987; Robinson and Osheroff, 1991). During DNA cleavage, topoisomerase I1 forms a covalent bond with the newly generated 5 ‘ nucleic acid termini (Liu et al., 1983; Sander and Hsieh, 1983). This covalent topoisomerase Il-cleaved DNA complex can be trapped in uitro by the addition of a protein denaturant such as sodium dodecyl sulfate (SDS) (Liu et al., 1983; Sander and Hsieh, 1983). The requirement for a protein denaturant obscured the mechanism of enzymemediated DNA cleavage for a number of years; it was not clear whether SDS trapped a normal reaction intermediate or rather induced DNA breakage within a noncovalent “precleavage” complex (Liu et al., 1983). Hence, the enzyme-DNA complex isolated by SDS treatment was originally termed the “cleavable complex” (Nelson et a / . , 1984) to reflect this mechanistic ambiguity. Recent studies. however, have characterized DNA substrates that undergo spontaneous topoisomerase II-mediated cleavage even in the absence of protein denaturants (Gale and Osheroff, 1990; Andersen et al., 1991). These results demonstrate that the coval-

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ent enzyme-cleaved DNA complex isolated in vitro corresponds to a normal cleavage intermediate in the catalytic cycle of topoisomerase 11. Accordingly, this complex is referred to as the “cleavage complex” for the remainder of this chapter.

C. Step 3: DNA Strand Passage Upon ATP binding topoisomerase I1 undergoes a structural reorientation (Lindsley and Wang, 1991). During this reorientation the passage helix is translocated through the transient double-stranded break made in the cleavage helix (Osheroff et al., 1983). It should be emphasized that ATP hydrolysis is not required for DNA strand passage.

D. Step 4: Post-Strand Passage DNA Cleavage/Religation Following the DNA strand passage event, the enzyme once again establishes a DNA cleavage/religation equilibrium (Osheroff, 1986; Robinson and Osheroff, 1991). Thus, topoisomerase I1 generates cleavage complexes both prior to and following its strand passage event. While the properties of these complexes and the kinetic pathway of their formation are similar, the post-strand passage cleavage complex is intrinsically fourfold more stable than its pre-strand passage counterpart (Osheroff, 1986; Robinson and Osheroff, 1991).

E. Step 5: ATP Hydrolysis \

As a prelude to enzyme turnover, topoisomerase I1 hydrolyzes its ATP cofactor to ADP and inorganic phosphate (Miller et al., 1981; Osheroff et al., 1983; Schomburg and Grosse, 1986). Little is known about the ATPase reaction of the type I1 enzyme. However, it is required for overall catalytic activity, is greatly stimulated by DNA, and is specifically inhibited by coumarin-based drugs such as novobiocin and coumermycin (Osheroff et al., 1983; Wang, 1985).

F. Step 6: Enzyme Turnover Enzyme turnover is the process by which topoisomerase I1 regains its ability to initiate a new round of catalysis. Although this reaction step remains almost a complete enigma, two important conclusions have emerged (Osheroff, 1986; Roca and Wang, 1993). First, enzyme recycling is dependent on ATP hydrolysis. Second, prior to turnover, topoisomerase I1 is unable to dissociate from the nucleic acid product of its doublestranded DNA passage reaction.

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IV. Enhancement of Topoisomerase Il-Mediated DNA Breakage by Antineoplastic Drugs The discovery that cells treated with Adriamycin or ellipticine contained high levels of protein-associated DNA breaks led Ross et al. (1978, 1979) to suggest that certain classes of antineoplastic agents acted by stabilizing covalent topoisomerase II-DNA complexes. Although further characterization of drug action in cells and isolated nuclei strongly supported this suggestion (Zwelling et al., 1981; Pommier et al., 1984, 1985a), the critical evidence linking topoisomerase II-mediated DNA breakage to drug action came from studies on the purified enzyme. The first such study was carried out by Nelson et al. (1984) and demonstrated that amsacrine (m-AMSA), but not its inactive o-isomer, dramatically stimulated the formation of topoisomerase II-DNA cleavage complexes. A number of subsequent studies by Liu and co-workers (Chen et al., 1984; Nelson et al., 1984; Twewy et al., 1984a,b) as well as Kohn and co-workers (Pommier et al., 1985b; Minford et al., 1986) drew similar conclusions for several antineoplastic drugs. In the relatively few years since the initial observations were made, an overwhelming body of literature (reviewed by Liu, 1989; Schneider et al., 1990; Corbett and Osheroff, 1993) has confirmed that the antineoplastic effects of many drug classes result from their ability to enhance topoisomerase II-mediated DNA breakage. However, the mechanism by which these agents corrupt the activity of the type I1 enzyme remains a subject of evolving debate. Historically, topoisomerase II-targeted compounds have been grouped into two broad mechanistic classes based on their interactions with DNA. Intercalative drugs were thought to enhance enzyme-mediated DNA breakage through their binding to the double helix, while nonintercalative agents [which originally were believed not to complex with DNA (Ross et al., 1984)l were thought to function by interacting directly with topoisomerase I1 (Liu, 1989; Schneider et al., 1990). However, subsequent findings that demonstrated binding of nonintercalative enzyme-targeted compounds to DNA argued for a more unified mechanism of action in which all antineoplastic drugs exert their effects at the enzyme-nucleic interface (Chow et al., 1988). Consistent with studies on gyrase- (Shen et al., 1989) or topoisomerase I-targeted (Hertzberg et al., 1989) agents, current models place topoisomerase II-targeted drugs in the ternary enzyme-DNA complex (Chow et al., 1988; Liu, 1989; Schneider et al., 1990; Corbett and Osheroff). However, evidence from a number of laboratories strongly suggests that a single encompassing mechanism for drug action is too simplistic. To further

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elaborate on this hypothesis, the following sections not only discuss the common features that link topoisomerase 11-targetedcompounds, but also point out mechanistic differences between drug classes.

V. Effects of Antineoplastic Drugs on the Sites of Topoisomerase II-Mediated DNA Breakage Topoisomerase I1 breaks the DNA backbone at preferred sites (Wang, 1985; Osheroff, 1989a; Osheroff et al., 1991). Although a large number of DNA cleavage sites have been mapped, no one consensus fits all of the known sequences. In fact, at least four different consensus recognition sequences for the enzyme have been proposed (Sander and Hsieh, 1983, 1985; Muller et al., 1988; Andersen el al., 1989; Capranico et al., 1990). Thus, the nucleotide determinants that provide specificity for the DNA cleavage reaction of topoisomerase I1 remain an open question. The above notwithstanding, the cleavage pattern generated by the enzyme on any given piece of DNA is remarkably reproducible. Even type I1 topoisomerases from widely divergent species produce similar patterns (Andersen et al., 1989). DNA cleavage patterns generated by topoisomerase I1 in the presence of most antineoplastic agents diverge dramatically from those obtained in the absence of drugs (Chen et al., 1984; Nelson et al., 1984; Tewey et al., 1984a,b; Riou et al., 1986; Markovits et af., 1989; Capranico et al., 1990; Sq5rensen et al., 1990; Pommier et al., 1991). At the very least, most antineoplastic agents significantly alter the site utilization of the enzyme (Chen et af., 1984; Nelson et al., 1984; Tewey et al., 1984a,b; Riou et al., 1986; Fosse et al., 1988 Markovits el al., 1989; Sgrensen et af., 1990; Pommier et al., 1991). Thus, while many sites of drug-promoted DNA breakage appear to be the same as those intrinsically recognized by topoisomerase 11, relative levels of DNA breakage at any particular site in the presence and absence of drug often differ widely. At least one antineoplastic agent, Adriamycin, has been shown to alter the intrinsic DNA cleavage specificity of the enzyme (Capranico et af., 1990).In a study that mapped 97 sites of topoisomerase 11-mediated nucleic acid breakage generated in the presence of Adriamycin and 90 sites generated in the absence of drug, the two classes of sites were found to be mutually exclusive. Finally, while most antineoplastic agents increase the promiscuity of topoisomerase I1 (greatly expanding the number of DNA sequences cleaved by the enzyme), two classes of drugs appear to act in a more prudish fashion. Although the benzisoquinolinedione amonafide (Hsiang et al., 1989) and the 2-nitroimidazole Ro 15-0216 (Sgrensen et al., 1990)

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both enhance overall levels of topoisomerase II-mediated DNA breakage, they do so by confining cleavage to a single site or a relatively small number of strong sites.

VI. Kinetic Pathway by Which Antineoplastic Drugs Enhance DNA Breakage Topoisomerase II-targeted drugs stabilize but do not trap DNA cleavage complexes (Liu, 1989; Schneider et al., 1990;Corbett and Osheroff, 1993). In other words, these compounds shift the DNA cleavageheligation equilibria of the enzyme toward the cleavage event. Accordingly, drug-induced enhancement of nucleic acid breakage can result from either an increase in the forward rate of enzyme-mediated DNA cleavage or an inhibition of the reverse religation reaction. Unfortunately, for a number of years, the tight coupling of DNA cleavage and religation proved to be a formidable hurdle to detailing the kinetic pathway of drug action. While it generally was assumed that antineoplastic agents enhanced topoisomerase IImediated DNA breakage by inhibiting religation (Liu, 1989; Schneider et al., 1990), it was impossible to address this critical point until assays that uncoupled religation from the cleavage reaction were developed. Recently, three independent assays specific for religation were reported. The first assay takes advantage of the finding that enzyme-DNA cleavage complexes established in the presence of calcium (rather than magnesium) can be trapped in a kinetically competent form following chelation of the divalent cation (Osheroff and Zechiedrich, 1987; Zechiedrich er al., 1989). The other two assays take advantage of the observation that the religation reaction of the enzyme is considerably less sensitive to extremes of temperature (either hot or cold) than is its DNA cleavage reaction (Liu er al., 1983; Osheroff and Zechiedrich, 1987; Hsiang and Liu, 1989; Hsiang et al., 1989; Robinson and Osheroff, 1991). All of these assays have been used to determine the apparent first-order rate of topoisomerase IImediated DNA religation in the absence or presence of antineoplastic drugs. Before discussing the kinetic pathway by which drugs stabilize DNA cleavage complexes, an important issue must be considered. As described above, topoisomerase I1 establishes DNA cleavage/religation equilibria both prior to and following its strand passage event (Osheroff, 1986; Robinson and Osheroff, 1991). Clearly, a complete analysis of drug action requires the effects of antineoplastic agents on both enzyme-DNA complexes to be assessed. Most studies on drug-induced DNA breakage are carried out in the presence of ATP. Unfortunately, since the high-energy

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cofactor supports enzyme turnover as well as strand passage (Osheroff, 1989a; Osheroff et al., 1991), the presence of ATP makes it impossible to attribute the effects of antineoplastic drugs on topoisomerase II-mediated DNA cleavage to any specific step in the catalytic cycle of the enzyme. Thus, while the inclusion of ATP in assays provides a model system that may more closely reflect the in vivo environment of DNA cleavage, it presents a stumbling block to the mechanistic analysis of drug action. However, since the DNA strand passage event catalyzed by topoisomerase I1 requires ATP binding but not hydrolysis (Osheroff er al., 1983), pre- and post-strand passage equilibria can be segregated from one another by utilizing a nonhydrolyzable ATP analog such as adenyl-5’-yl imidodiphosphate [APP(NH)P] in reactions (Osheroff, 1986;Robinson and Osheroff, 1991). In the absence of an ATP cofactor, only pre-strand passage cleavage complexes are generated. However, in the presence of APP(NH)P 2 75% of the complexes observed are post-strand passage in nature (Robinson and Osheroff, 1991).

A. Effects of Antineoplastic Drugs on the Pre-Strand Passage DNA Cleavage/Religation Equilibrium In the absence of ATP, antineoplastic drugs enhance pre-strand passage DNA breakage mediated by topoisomerase I1 (Chen er al., 1984; Pommier et al., 1985b; Osheroff, 1989b; Robinson and Osheroff, 1990, 1991; Robinson et al., 1991). For example, etoposide increases levels of doublestranded DNA breaks generated by the Drosophila type I1 enzyme as much as 10-fold (Osheroff, 1989b; Robinson and Osheroff, 1991). The DNA religation assays described above have been used to determine whether etoposide or amsacrine impairs the ability of Drosophila topoisomerase I1 to rejoin cleaved nucleic acids prior to strand passage (Osheroff, 1989b; Robinson and Osheroff, 1990; Robinson et al., 1991). Both antineoplastic drugs decreased apparent first-order rates of DNA religation. Furthermore, the degree of inhibition observed correlated with levels of drug-enhanced nucleic acid breakage. Therefore, two structurally disparate classes of topoisomerase II-targeted agents appear to enhance pre-strand passage DNA breakage primarily by impairing the religation of cleaved nucleic acid molecules. One further result concerning the actions of antineoplastic agents on DNA religation must be noted. In order for either etoposide or amsacrine to inhibit DNA religation, drug had to be present at the time of the cleavage event (Osheroff, 1989b; Robinson and Osheroff, 1990). This finding suggests that these compounds act within the ternary enzyme-DNA complex.

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B. Effects of Antineoplastic Drugs on the Post-Strand Passage DNA Cleavage/Religation Equilibrium As described above, the post-strand passage DNA cleavage complex of topoisomerase I1 [characterized in the presence of APP(NH)P] is approximately fourfold more stable than its pre-strand passage counterpart (Osheroff, 1986; Robinson and Osheroff, 1991). Moreover, the enhanced stability that occurs following strand passage reflects a decrease in the rate of DNA religation (Robinson and Osheroff, 1991). Thus, as might be expected, antineoplastic drugs have a less pronounced influence on topoisomerase 11-mediated DNA breakage that follows the strand passage event. Although etoposide and amsacrine enhance post-strand passage DNA cleavage, their relative effects are two- to threefold lower than those observed pre-strand passage. However, since levels of post-strand passage DNA cleavage complexes formed in the absence of drugs are three- to fivefold higher than those observed before strand passage, considerable nucleic acid breakage is induced by drug treatment. The effects of etoposide and amsacrine on the post-strand passage DNA religation activity of Drosophila topoisomerase I1 have been determined (Robinson and Osheroff, 1991). Either drug caused substantial reductions (two- to 10-fold) in the apparent first-order rate of post-strand passage DNA religation. Two conclusions concerning topoisomerase 11-targeted drugs can be drawn from these studies. First, DNA cleavage complexes established both prior to and following strand passage appear to be targets for either etoposide or amsacrine. Second, as found for pre-strand passage events, these compounds stabilize post-strand passage DNA cleavage complexes primarily by inhibiting the religation reaction of the enzyme. As a note of concern, caution should be applied when extrapolating the above conclusions to other classes of topoisomerase 11-targeted agents. While the enhancement of nucleic acid breakage is common to all drug classes, the inhibition of DNA religation is not a universal feature of drug mechanism. A case in point is the quinolones, whose kinetic pathway of action is in such contrast to etoposide and amsacrine that they must be considered as a separate mechanistic class of compounds.

C. Quinolones as a Novel Mechanistic Class of Topoisomerase 11-Targeted Drugs Of all the agents targeted to type I1 topoisomerases, quinolone-based drugs are perhaps the most widely used for the treatment of human disease (Zimmer et af., 1990; Hooper and Wolfson, 1991). However, in contrast

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to the compounds discussed above, quinolones are used exclusively as antimicrobial agents targeted to DNA gyrase [the prokaryotic counterpart of topoisomerase I1 (Sutcliffe et al., 1989; Reece and Maxwell, 1991; see also Zimmer et al., 1990; Hooper and Wolfson, 19911. In fact, the 6fluoroquinolone ciprofloxacin is the most active oral antibiotic currently in clinical use (Zimmer et a / . , 1990; Hooper and Wolfson, 1991). At concentrations well above their therapeutic ranges, many clinically relevant quinolones inhibit the overall catalytic activity of topoisomerase I1 (Miller et al., 1981; Osheroff et al., 1983; Hussy et al., 1986; Oomori et al., 1988; Hoshino et al., 1989, 1991; Gootz et al., 1990; Moreau et al., 1990). Furthermore, the most potent members of this antimicrobial drug class, such as ciprofloxacin, are weak enhancers of DNA breakage mediated by the eukaryotic enzyme (Barrett et al., 1989). Recently, quinolones with far greater activity toward eukaryotic topoisomerase I1 have been described (Barrett et al., 1989; Jefson et al., 1989; Gootz et al., 1990; Robinson et al., 1991, 1992; Wentland et al., 1991, 1992; Kohlbrenner et al., 1992; Yamashita et al., 1992). One such compound, CP-115,953 (see Fig. l), displays the highest activity toward the eukaryotic enzyme of any quinolone reported to date and is the first quinolone found to be more potent than a widely used antineoplastic drug such as etoposide (Robinson et al., 1991). As seen in Fig. 3 (left panel), CP-l15,953 is a potent enhancer of prestrand passage DNA breakage mediated by Drosophila topoisomerase I1 (Robinson et al., 1991). The quinolone shows an even higher activity against the mammalian type I1 enzyme (Robinson ef al., 1991; Elsea et al., 1993). The relative potency of CP-115,953 (compared to etoposide) observed for post-strand passage DNA breakage is similar to that found for the pre-strand passage reaction (Robinson et al., 1991). These results strongly suggest that at least some members of the quinolone family should be classified as topoisomerase II-targeted drugs. To determine whether quinolones share a common kinetic pathway of action with drugs such as etoposide or amsacrine, the effects of CP-115,953 (and related compounds) on topoisomerase II-mediated DNA religation were examined (Robinson et al., 1991). Results of pre-strand passage assays are displayed in Fig. 3 (right panel). In marked contrast to etoposide, the quinolone shows little ability to inhibit the pre-strand passage DNA religation reaction of the enzyme. In addition, CP-l15,953 does not inhibit (and if anything enhances) rates of DNA religation generated by topoisomerase I1 post-strand passage (Robinson et al., 1991). Therefore, CP-115,953 and related quinolones appear to represent a novel mechanistic class of topoisomerase II-targeted drugs. While agents such as etoposide and amsacrine increase levels of enzyme-DNA cleavage complexes pri-

k’^

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Fig. 3 Effects of the quinolone CP-I 15,953 on the DNA cleavage (left) and religation (right) reactions of Drosophifa topoisomerase 11. (Left) Levels of double-stranded DNA cleavage were determined by the conversion of negatively supercoiled plasmid pBR322 to linear molecules and were assigned a value of 1 in the absence of drug. A DNA cleavage titration generated in the presence of etoposide is shown for comparison. (Right) DNA religation was induced by a rapid shift in temperature from 30°C to 55°C (Robinson ef a/., 1991). Rates of DNA religation were monitored by the loss of linear plasmid molecules. Results obtained with 50 p M CP-115,953 are compared to those obtained in the absence of drug or in the presence of 100 p M etoposide.

manly by inhibiting DNA religation, the quinolones apparently do so by enhancing the forward-rate DNA cleavage. A recent study with Ro 150216, a nitroimidazole derivative that bears no structural resemblance to the quinolones, suggests that this drug also stabilizes cleavage complexes with little effect on rates of DNA religation (SZrensen et al., 1990). Thus, the ability to enhance topoisomerase 11-mediated DNA breakage without impairing religation may not be limited to the quinolones.

VII. Enzyme Interaction Domains for Topoisomerase II-Targeted Drugs The domain(s) on topoisomerase I1 that interacts with antineoplastic drugs has yet to be identified. However, point mutations in four independent isolates of topoisomerase I1 that display drug resistance have been mapped. They are the conversion of Arg-486 to Lys in the HLdO/AMSA and KBM-3/AMSA human enzymes (Hinds et al., 1991; Lee et al., 1992), Arg-449 to Glu in the CCRF-CEM human enzyme (Bugg et al., 1991), and Arg-493 to Glu in the VpmR-5Chinese hamster ovary enzyme (Chan

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et al., 1992). These results indicate that the region of topoisomerase I1 that is homologous to the B subunit of DNA gyrase (Wyckoff et af.,1989; Huang, 1990) probably is important for interactions with antineoplastic agents. Mutagenesis experiments have led to similar conclusions for the interaction of quinolones and/or antineoplastic drugs with DNA gyrase (Yamagishi et al., 1986; Reece and Maxwell, 1991; Yoshida et al., 1991) and bacteriophage T4 topoisomerase I1 (Huff et al., 1989). It is notable, however, that the A subunit of DNA gyrase as well as the corresponding subunit of the bacteriophage enzyme also appear to be quite important for drug action (Fisher et al., 1989; Huff et al., 1990; Yoshida et af., 1990; Hallett and Maxwell, 1991; Reece and Maxwell, 1991). A number of studies strongly suggest that different classes of topoisomerase 11-targeted drugs have overlapping but distinct interaction domains on the enzyme. First, the HL-601AMSA enzyme is resistant to a number of intercalative drugs, but retains sensitivity to nonintercalative agents such as etoposide (Zwelling et al., 1989, 1991). Second, the VpmR-5enzyme is resistant to most topoisomerase 11-targeted agents, yet is highly sensitive to quinolones (Robinson et al., 1991, 1992). Finally, while genistein, amsacrine, and the quinolone CP-115,953 all inhibit ATP hydrolysis catalyzed by topoisomerase 11, etoposide shows little ability to impair this important enzyme function (M. J. Robinson and N. Osheroff, unpublished observations).

VIII. Possible Ramifications of Mechanistic Diversity among Topoisomerase Il-Targeted Drugs

The mechanistic diversity among topoisomerase 11-targeted drugs has a number of potential cellular consequences. First, different antineoplastic agents produce distinct alterations in the DNA cleavage pattern generated by the enzyme. Since the lethal processing of drug-stabilized topoisomerase 11-DNA cleavage complexes is exacerbated by DNA replication or transcription (Liu, 1989; D’Arpa et af., 1990; Schneider et al., 1990), a compound that induces nucleic acid breaks preferentially in the vicinity of replication origins or actively transcribed genes may have a greater cytotoxic potential than a compound that induces breaks in less active regions of the genome. Second, the kinetic pathway by which drugs increase DNA breakage is not common among all topoisomerase 11-targeted agents. While a compound that inhibits DNA religation may be more clastogenic in nature, a compound that enhances the forward rate of DNA cleavage may be more likely to promote intermolecular ligation or other

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recornbinogenic events. Finally, drug interaction domains on topoisomerase I1 do not appear to be identical for all antineoplastic agents. This finding implies that a cancer that is resistant to one class of topoisomerase 11-targeted drugs due to a mutation in the enzyme may respond to treatment with another class of compounds.

IX. Perspectives and Conclusions All topoisomerase 11-targeted antineoplastic drugs described to date act by converting the enzyme into a cellular poison. Fundamental to their chemotherapeutic action is the ability to stimulate DNA breakage mediated by the type I1 enzyme. However, beyond this common denominator, it is clear that different classes of drugs act with individual personalities, leaving behind distinct mechanistic signatures. With this in mind, it is evident that the historical categorization of topoisomerase 11-targeted agents simply on the basis of their DNA intercalation properties is no longer a sufficient means of classifying different drug types. The challenge of the future is to exploit the mechanistic differences between drug classes in order to develop chemotherapeutic regimens that maximize the potential of topoisomerase I1 as a target for the treatment of human cancers.

Acknowledgments Work in the laboratory of the senior author (N.O.) has been supported by National Institutes of Health (NIH) grant GM33944 and by American Cancer Society Faculty Research Award FRA-370. A.H.C. and M.J.R. were trainees under NIH grant CA09582. We are grateful to J. Rule for expert assistance with photography and S. Heaver for conscientious preparation of the manuscript.

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