SEPTEMBERIOCTOBER 1993 VOLUME 6, NUMBER 5 0 Copyright 1993 by the American Chemical Society
Invited Review When Good Enzymes Go Bad: Conversion of Topoisomerase I1 to a Cellular Toxin by Antineoplastic Drugs Anita H. Corbett and Neil OsherofP Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received May 5,1993
Introduction Topological relationships in DNA such as over/underwinding, knotting, or tangling profoundly influence the cellular functions of the genetic material (I). Consequently, it is not surprising that topoisomerases, the enzymes that modulate the topological state of nucleic acids in vivo, play fundamental roles in all aspects of DNA metabolism (I). All eukaryotic cells contain two classes of topoisomerases. Members of these classes are referred to as type I or type I1 topoisomerases and can be distinguished on the basis of their physical and mechanistic properties (1-4). All known type I topoisomerases are monomeric in their active form (5-3,require no high-energy cofactor (I, 3), and alter nucleic acid topology by passing a single strand of DNA through a transient single-stranded nick made in the opposing strand (2,8, 9). In contrast, type I1 topoisomerases function as homodimers (7,10-14), act at the expense of ATP (IO,14,15),and alter DNA topology by passing an intact double helix through a transient double-stranded break made in a second helix (1,2,10,16, 17). While topoisomerase I is not essential for the viability of single-celled eukaryotes (18-201, it is required for embryogenesis in Drosophila (21). The enzyme is im-
* To whom correspondence should be addressed, at the Department of Biochemistry,621Light Hall,VanderbiltUniversitySchoolofMedicine, Nashville, T N 37232-0146;(615)322-4338(phone);(615)322-4349(FAX).
plicated most heavily in DNA processes such as transcription (1,2,22-27) and replication (28-31)that depend on the action of a swivel to alter the superhelical density of the genetic material. Consistent with its cellular role, topoisomerase I is associated with chromatin (32,33), is and appears to be expressed enriched in the nucleolus (23), constitutively throughout the cell cycle (34-36). Topoisomerase I1 is essential for the survival of all eukaryotic cells (19,37-41). I t plays important roles in DNA replication (29-31,42,43) and recombination (4449) and is required for the proper structure (50-53), condensationldecondensation,(41,54-59), and segregation (19,37,3+41,48,60) of chromosomes. Furthermore, levels of topoisomerase I1 increase substantially during periods As a reflection of rapid cell proliferation (34,36,61-65). of ita physiological functions, the type I1 enzyme is a major component of the nuclear matrix in interphase cells (66) and is the major polypeptide of the chromosome scaffold in mitotic cells (50-53). Finally, the enzyme is regulated over the cell cycle with content and activity peaking at GdM (35,67). For a number of years following their discoveries (16, 17,68-701,the type I and type I1topoisomerases remained within the sole domain of the biochemist and the molecular biologist. However, with the discovery that these enzymes were cellular targets for a number of clinically important antineoplastic agents (71-741,the scope of interest in topoisomerases rapidly expanded to include both the pharmacologist and the oncologist. The present review
0893-228~/93/2706-0585$04.00/0 0 1993 American Chemical Society
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586 Chem. Res. Toxicol., Vol. 6, No. 5, 1993
GXXGXG 161-166
GXGXXG 472-477
y805
Corbett and Osheroff tCOOH
Phosphorylation Sites
gyrA Homology Regulatory gyr5 Homology Domain Domain Domain 1. Domain structure of topoisomerase 11. The
Figure ATP binding motifs (residues 161-166 and 472-477) and the active site tyrosine involved in DNA cleavage (residue 805) are shown. Sequence positions correspond to those in the human type I1 enzyme (76) as amended by Hinds et ai. (77). will focus on the role of the type I1 enzyme in mediating drug-induced cytotoxicity as well as the mechanism by which antineoplastic agents alter topoisomerase 11activity. As detailed below, the topoisomerase II-targeted drugs act in a unique fashion. Rather than blocking the critical physiological functions of this enzyme, these drugs act by converting the essential type I1 enzyme into a potent cellular toxin. Before the actions of topoisomerase IItargeted drugs can be fully appreciated, however, it is necessary to have an understanding of the enzyme and how it carries out its catalytic function. Therefore, the following sections will provide a brief introduction to topoisomerase 11.
Topoisomerase I1 As described above, topoisomerase I1 alters DNA topology by a double-stranded DNA passage reaction ( I , 2). As a consequence of its reaction mechanism, the type I1enzyme is able to remove positive or negative superhelical twists from the genetic material and resolve intramolecular DNA knots as well as intermolecular nucleic acid tangles (1, 2). On the basis of amino acid sequence comparisons with the prokaryotic type I1 enzyme, DNA gyrase (75),topoisomerase I1 can be divided into three distinct domains (Figure 1) (78,791.l The N-terminaldomain is homologous to the B subunit of gyrase and contains consensus sequences for ATP binding (80,8I). The central domain is homologous to the A subunit of gyrase and contains the tyrosine residue that forms the covalent linkage with DNA during the scission reaction (82). The C-terminal domain is highly variable and has no corresponding region of homology in gyrase (78,79). It contains a number of sites that are phosphorylated in vivo (83) and is postulated to play a role in the physiological regulation of the enzyme (83, 84). For a number of years, only a single isoform of topoisomerase I1was believed to exist in eukaryotic species. As of this writing, this still appears to be the case for lower eukaryotes such as yeast (38,85-87) and Drosophila (79, 88). However, recent studies have demonstrated the existence of a second isoform of the enzyme in mammals (89,90). The two isoforms have been designated CY and (3 (90) and are distinguished by their polypeptide molecular masses 1-170 kDa for a us -180 kDa for j3 (89)l. The a isoform is the type I1 enzyme originally described in ‘AU amino acid sequencedesignationscorrespondto positions in human topoisomerase 11. The sequence originally reported in ref 76 contained 1530 amino acids. The amended sequence reported by Hinds et al. (77) contains one additional amino acid in the region of residues 109-114. The correction in the primary structure of the enzyme has been confirmed by Jenkina et al. (93). Thus, the sequence positions given in this review correspond to those of the amended 1531 amino acid human type I1 enzyme.
mammals (10) and appears to be the only type I1 enzyme found in lower eukaryotes (7,11,12,91). Topoisomerase IIa and j3 share extensive (-70%) amino acid identity (90-93). If the nonconserved C-terminal domain is excluded from homology comparisons, sequence identity rises to -80% (91,93). Despite their similarities, the a and j3 isoforms are encoded by separate genes that have distinct chromosomal locations (17q21-22 and 3p24 for the human a and (3 genes, respectively) (76, 90,93, 94). To date, the overwhelming majority of in vitro studies on topoisomerase I1 have focused on the a isoform ( I , 2, 95). Although enzymological studies on j3 have been severely limited, no significant mechanistic differences between the two isoforms have been identified (96). Many of the in vivo studies on topoisomerase I1function and regulation specifically manipulated the gene that encodes the a polypeptide (19, 37-41, 48) or utilized antibodies that were specific for the a isoform of the enzyme (35, 50-53, 62, 66). Thus, it is likely that topoisomerase IIa is the isoform most intimately associated with DNA/chromosome metabolism. This suggestion is supported by the finding that a is the predominant isoform found in most proliferating tissues (97-100) and that j3 appears to be confined primarily to the nucleolus (101). Although in vitro the a isoform of topoisomerase I1 appears to be more sensitive to antineoplastic agents than /3 (96,97),most in vivo studies that utilized mammalian systems have not determined the specific contributions of the two isoforms to the pharmacological effects of drugs. Since much of what is known concerning the mechanism of action of topoisomerase II-targeted drugs and the catalytic mechanism of the enzyme comes from studies with species that contain only a single form of the enzyme, this review will make no further attempt to distinguish between the two isoforms. Therefore, the enzyme will be referred to simply as topoisomerase I1 for the remainder of this review.
Catalytic Cycle of Topoisomerase I1 Studies carried out over the past decade have separated the double-stranded DNA passage reaction of topoisomerase I1 into a number of discrete steps. These are shown in Figure 2, which depicts one round of the enzyme’s catalytic cycle (see ref 95 for a recent review). A brief description of each reaction step follows. (1) Topoisomerase I1 binds to ita DNA substrate at points of helix-helix juxtaposition (103, 104). Binding takes place at preferred nucleic acid sequences (105,206) and requires no cofactors (107, 108). (2) In the presence of a divalent cation, topoisomerase I1 establishes a DNA cleavage/religation equilibrium ( I I , 107,109). The enzyme cuts the genetic material within its DNA recognition sequence (11,110-113)and generates double-stranded 5’-phosphate/3’-hydroxylbreaks that contain four-base 5’-overhangs (11, 109). During the scission reaction, topoisomerase I1 maintains the topological integrity of its nuleic acid substrate by forming a covalent bond with both newly formed 5’-DNA termini via an 04-phosphotyrosyl linkage (82, 114). Due to the transient nature of this covalent interaction between the enzyme and double-stranded DNA, the addition of a protein denaturant such as sodium dodecyl sulfate is required to disrupt the cleavage/religation equilibrium in vitro and trap topoisomerase I1 in ita DNA cleavage complex(11,109,115). Although theabilityoftheenzyme
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x+m f
\4
2-/
Mg2+
ATP Figure 2. Catalytic cycle of topoisomerase I1 (95). The homodimeric enzyme is represented by the handlebar-shaped structure, and the DNA helices are represented by the cylinders. The change in enzyme structure that takes place following step 3 represents the structural transition that occurs upon ATP binding (102). The double-stranded DNA passage reaction of topoisomerase I1 can be broken into six discrete steps: (1) enzymeDNA binding; (2) pre-strand passage DNA cleavage/ religation; (3) double-stranded DNA passage; (4)post-strand passage DNA cleavageheligation; (5)ATP hydrolysis;(6) enzyme turnover. Transient enzymeDNA cleavagecomplexesare shown in brackets.
to cleave and religate its DNA substrate is fundamental to its catalytic function, as discussed later in this review, perturbation of this critical DNA cleavageheligation equilibrium by antineoplastic drugs converts topoisomerase I1 from an essential enzyme to a cellular toxin. (3) Upon binding of its ATP cofactor, topoisomerase I1 undergoes a structural transition (102). Concomitant with this transition is the transport of an intact DNA helix through the transient double-stranded DNA break described in step 2 (15, 116, 117). This ATP-induced conformational change in the enzyme causes topoisomerase I1 to become topologically linked to its nucleic acid substrate, forming a “sliding clamp” that can move along DNA by one-dimensional diffusion but cannot dissociate from circular substrates (116, 117). (4) Following the DNA strand passage event described in step 3, the enzyme once again establishes a DNA cleavage/religation equilibrium (116, 118). While the kinetic pathway that leads to the formation and disappearance of the post-strand passage topoisomerase IIDNA cleavage complex is comparable to that of its prestrand pwage counterpart, the cleavage complex generated following strand passage is intrinsically 2-4 times more stable than that generated prior to ATP binding (116, 118). ( 5 ) Topoisomerase I1 hydrolyzes its ATP cofactor to ADP and orthophosphate (10, 14, 15, 119). (6) The ATP hydrolysis reaction of step 5 triggers
regeneration of the pre-strand passage conformation of the enzyme, allowing topoisomerase I1 to dissociate from its DNA product and initiate a new round of catalysis (i.e., enzyme turnover) (116).
Regulation of Topoisomerase I1 by Phosphorylation Topoisomerase I1 exists in the cell as a phosphoprotein (83, 120-126). Modification appears to be confined
primarily to serine residues; however, low levels of phosphothreonine also have been reported (83,121,123,124,
126). Phosphorylation of topoisomerase I1 appears to be cell cycle regulated with modification peaking at the G2/M boundary (83,122). Thus far, casein kinase I1 is the only kinase that has been demonstrated to play a role in the physiological phosphorylation of the enzyme (83,121,127). Although studies with cellular enhancers and inhibitors of protein kinase C have implicated an in vivo role for this kinase, confirming evidence for a physiological interaction between protein kinase C and topoisomerase I1 has yet to be established (120, 128-130). In vitro, topoisomerase I1 is a high-affinity substrate for casein kinase I1 (83,131-1341, protein kinase C (83,84, 120, 133, 135), calcium/calmodulin-dependent protein kinase (134,135),and p34dC2 kinase (83,134). Phosphorylation by any of these kinases enhances catalytic activity from -3- to >20-fold depending on the species of type I1 enzyme used (84,120,131-135). As determined by studies on Drosophila topoisomerase I1 (84,132,1331, both casein kinase I1 and protein kinase C enhance catalytic activity specificallyby increasingthe rate of enzyme-mediatedATP hydrolysis (step 5 in the catalytic cycle of the enzyme) and presumably the rate of enzyme turnover. The mechanism by which phosphorylation stimulates the ATPase activity of the enzyme is not obvious. However, it has been suggested that the C-terminal domain of topoisomerase I1 (see Figure 1) is autoinhibitory in nature and is neutralized by phosphorylation (84, 127).
Topoisomerase II-Targeted Antineoplastic Drugs Beyond its essential physiological functions, topoisomerase 11is the primary cellular target for a wide variety of antineoplastic drugs (71-74). Many of these agents are listed in Table I, and structures for selected compounds are shown in Figure 3. As can be seen, agents targeted to topoisomerase I1are derived from anumber of structurally diverse drug classes. One of the few properties shared by all drugs examined to date is the ability to bind to DNA (71-73,148). However,even within this common property, there is considerable divergence; while compounds such as etoposide and genistein are nonintercalative in nature (147,149,160), others such as doxorubicin and amsacrine intercalate strongly into the genetic material (136-138, 144) (see Table I). The contribution of DNA binding to the activity of topoisomerase II-targeted agents is not welldefined. However, on the basis of studies that characterized the mechanism of action of drugs targeted to DNA gyrase (165,166) or the eukaryotic type I enzyme (167), it is likely that the site of action of compounds targeted to eukaryotic type I1 topoisomerase is the enzyme-DNA complex. Many of the compounds shown in Figure 3 are routinely used for the treatment of human cancers (see Table I). For example, when administered in combination chemotherapy, etoposide is an effective agent against germ line cancers, non-Hodgkins lymphomas, and several leukemias (145, 150, 153). Furthermore, in single-agent regimens, this compound has shown promise as a therapy for smallcell lung carcinomas (151, 152). Although a number of the drugs listed in Table I are still confined to laboratory usage, all of the compounds tested display activity against mammalian cellsand/or tumor models (159,163,164,168). Of particular note in this latter category are the quinolonebased agents. Members of this drug class include some of the most active antimicrobial agents currently in clinical
688 Chem. Res. Toxicol., Vol. 6,No.5, 1993
Corbett and Osheroff
Table I. Topoisomerase 11-Targeted Drugs examples DNA binding mode Drugs with Clinical Applications amsacrine intercalative mitoxantrone intercalative doxorubicin intercalative daunomycin etoposide nonintercalative teniposide Drugs Currently in Clinical Trials amondide intercalative ellipticine intercalative 2-methyl-9-hydroxyellipticine Drugs Currently in Laboratory/Experimental Use genistein noninbrcalative RO 15-0216 nonintercalative CP-115,953 nonintercalative
drug class anilinoacridines anthracenediones anthracyclines epipodophyllotoxins benzoisoquinolinediones ellipticines isoflavones nitroimidazoles quinolones H3C
-+
0
OH
HICO 0 = ? H
OH
refs 136-139 140-143 144-146 145,147-153
154-156 141,157,158
159-161 162 163,164
0
H,c@ NH,'
I
H
I
CH,
H,S
Ellipticine
Amonafide
CP-115,953
Cenistein
OH
Doxorubicin I
ai
Etoposide OH
0
HN(CHz)INH(CHzhOH
OH
0
HN(CHz)2NH(CH&OH
#$)
C H 3 O Y
&
Mitoxantrone Amacrine Figure 3. Structures of selected topoisomerase 11-targeted drugs.
use (169,170).While the antimicrobial quinolones [which are targeted to DNA gyrase (75,171)lshow little activity against the eukaryotic type I1 enzyme (172,173))derivatives of these compounds are potent effectors of the eukaryotic enzyme and display antineoplastic potential (163,164,172-179).
In Vivo Effects of Topoisomerase 11-Targeted Agents Typically, the efficacy of cytotoxic drugs reflects their ability to inhibit the actions of an essential enzyme or to block a critical cellular process. An example of such a drug is methotrexate, a compound with a considerable history as an antineoplastic agent (180).Methotrexate is a potent inhibitor of dihydrofolate reductase, an enzyme required for the regeneration of tetrahydrofolate from dihydrofolate and hence the conversionof dUMP to dTMP (181,182). By interfering with this essential enzymecatalyzed event, the drug effectively blocks the synthesis of DNA (182).In many cases, cells that display high levels of resistance to methotrexate have undergone a spontaneous amplification of the gene locus that encodes dihydrofolate reductase (183).Thus, consistent with the mechanism of methotrexate action, the cytotoxic effects of this drug can be overcome by increasing the cellular concentration of its enzyme target. The topoisomerase 11-targeted agents stand in marked contrast to drugs like methotrexate. While at high
RO 15-0216
concentrations, the compounds described in Table I inhibit the overall catalytic activity of the type I1 enzyme (138, 141,144,147,159,162,1841,the cytotoxic potential of these drugs correlates with their ability to stabilize covalent complexes between topoisomerase I1 and cleaved DNA (71-74).As described above, these enzyme-DNA cleavage complexes are fleeting intermediates in the doublestranded DNA passage reaction of topoisomerase I1 (see Figure 2) (2,185,186). Cleavage complexes are normally present in low concentrations in untreated cells. However, following treatment with topoisomerase 11-targeted drugs, the cellular concentration of these enzyme intermediates increases dramatically (187-192).Although the nucleic acid breaks present in cleavagecomplexes are transient in nature, many are subsequently converted to permanent breaks when replication complexes (and to a lesser extent, transcription complexes) attempt to traverse these proteinaceous roadblocks in the DNA (73,193-195). Hence, cells that are treated with topoisomerase 11-targetedagents contain high levels of protein-associated double-stranded breaks in their genetic material (187-192).The presence of these DNA breaks greatly stimulates nucleic acid recombination/ mutation (196-199)and the formation of chromosomal abnormalities (200-204)and eventually triggers cell death by the process of apoptosis (205-208). Thus, the drugs listed in Table I are insidious in nature; they do not destroy the ability of the type I1 enzyme to mediate ita essential
Invited Reviews
cellular functions but rather subvert the unwitting enzyme to a potent physiological toxin. The hypothesis that drugs convert type I1 topoisomerases into cellular toxins was originally proposed by Kreuzer and Cozzarelli (209)to explain the mechanism of action of quinolone-basedantimicrobials targeted to DNA gyrase. The first suggestion that certain classes of antineoplastic drugs killed eukaryotic cells by stabilizing covalent topoisomerase11-DNA cleavage complexes came from the observation by Ross et al. (210, 211) that eukaryotic cells treated with adriamycin or ellipticine contained high levels of protein-associated breaks in their DNA. Since these pioneering studies, an overwhelming body of evidence has confirmed that the type I1 enzyme is the primary physiological target for a number of antineoplastic agents and that these drugs act by transforming topoisomeraseI1into a cellular toxin. First, there is a high correlation between the ability of compounds to stabilize cleavage complexes in vitro and to kill cells in Second, vivo (71-74,138,147,149,163,172,178,184,212). many mutant cell lines that display high levels of resistance to antineoplastic agents express mutant forms of topoisomerase I1 (77,213-228).2 Furthermore, the profiles of drug resistance exhibited by these mutant enzymes in vitro are similar to those displayed by the mutant cell lines in vivo (213-223).3Third, sensitivity to antineoplastic agents parallels cellular levels of topoisomerase I1 (62,64,65,67). The most dramatic examplesof this correlation come from studies on genetically altered yeast. While yeast cells that have been engineered to overexpress topoisomerase I1show significant increases in their sensitivity to drugs (229), cells that have been engineered to contain low levels of topoisomerase I1 activity are nearly refractory to the cytotoxic effects of these agents (198,230). Since rapidly proliferating cells contain elevated levels of the type I1 enzyme (34,36,61-65),the lethal effects of topoisomerase11-targeted agents are most pronounced in fast-growing or in neoplastic tissues (34, 64, 224, 231236). This is one of the major reasons why cancerous cells, especially those of an aggressive nature, are more susceptible to the adverse effects of these compounds than are normal tissues. Finally, a topic of current debate concerns the potential role of phosphorylation as a modulator of drug sensitivity. In vitro, modification of topoisomerase I1by either casein kinase I1 or protein kinase C slightly decreases (up to 35% ) the stimulation of enzyme-mediated DNA cleavage by etoposide or amsacrine (133). However, results from in vivo studies are considerably more complex. While topoisomerase I1 is hyperphosphorylated in two mutant cell lines that display resistance to etoposide (237), the enzyme is 2- to %fold underphosphorylated in an amsacrine-resistant mutant line (238). Thus, for the time being, the relationship between topoisomerase I1phosphorylation and drug sensitivity remains an open question.
-
Mechanism of Action of Topoisomerase 11-TargetedDrugs As discussed above, the antineoplastic drugs listed in Table I act by enhancing topoisomerase11-mediatedDNA 2It should be noted that, to date, all of the mutant drug-resistant mammalian type I1 topoisomerases that have been described appear to be of the CY isoform. 3D.M. Sullivan, M. D. Latham, M. J. Robinson, and N. Osheroff, unpublished observations.
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 589
FIIFIIIC
I
PI-
*
Figure 4. Stimulation of topoisomerase 11-mediated DNA cleavage by antineoplastic drugs. An ethidium bromide-stained agarose gel is shown. Assays were carried out as previously described (163) and contained 100 nM topoisomerase I1 and 5 nM negatively supercoiled pBR322 plasmid DNA. DNA cleavage was carried out in the presence of no drug or 100 pM amsacrine (AMSA),etoposide (Etop), genistein (Gen),or CP-115,953(953). The positions of negativelysupercoiled (form I, FI),nicked (form 11,FII), and linear (form 111,FIII) DNA molecules are indicated.
breakage (71-74). Results of a typical DNA cleavage assay are shown in Figure 4. As monitored by the conversion of negatively supercoiled plasmid DNA (form I, FI) to linear molecules (form111,FIII),topoisomerase11-targeted drugs stimulate double-stranded DNA breakage 5- to IO-fold. The global stimulation of DNA scission induced by the presence of drugs is not uniformly distributed among all topoisomerase I1 recognition/cleavage sites. While DNA breakage at some sites increases dramatically (1-2 orders of magnitude), breakage at other sites is often unaffected (138,141,144,147,239-241). Furthermore, the pattern of site utilization by the enzyme appears to be dependent on the drug class employed. Relationships (if any) between the site specificity and cytotoxicity of these compounds have yet to be explored (242). The enhancement of DNA breakage by topoisomerase 11-targeted agents can be accomplished by two different but not mutually exclusive mechanisms. Drugs may act by stimulating the forward rate of enzyme-catalyzedDNA cleavage or by decreasing the rate of DNA religation. For a number of years, the mechanism of action of topoisomerase 11-targetedagents was obscured by the transient nature of the covalent enzyme-DNA cleavage complex and by the tight coupling of the DNA cleavage and religation reactions. While it was generally assumed that all topoisomerase 11-targeted drugs acted by impairing the ability of the enzyme to rejoin cleaved DNA (71-73), it was impossibleto address this critical point until assays that uncoupled religation from the DNA cleavage/religation equilibrium were developed. In recent years, four independent assays specific for religation have been reported. Two of these assays take advantage of the observation that the DNA cleavage reaction of topoisomerase I1 is considerably more sensitive to extremes of temperature than is its DNA religation reaction (109,116, 118, 154, 243). The other two assays utilize alternative divalent cations (either Ca2+or Mn2+in place of Mg2+) (115, 244) or suicide DNA substrates (245) to trap the covalent enzyme-DNA cleavage complex in a kinetically competent form. Results of DNA religation assays demonstrate that topoisomerase 11-targeted antineoplastic agents fall into two distinct mechanistic classes. A summary of results for the drugs examined to date is shown in Figure 5. Some drugs, such as etoposide, severely inhibit enzyme-mediated DNA religation (118, 246-248). This finding strongly suggests that one mechanistic class of drugs acts primarily
-
590 Chem. Res. Toxicol., Vol. 6, No. 5, 1993 Quinolones Genistein Nitroimidazoles
Etoposide Amsacrine
Figure 5. Mechanism by which antineoplastic drugs enhance topoisomerase II-mediated DNA breakage. The enzyme is represented by the handlebar-shaped structure, and the DNA helices are represented by the cylinders. The transient covalent topoisomerase II-cleaved DNA complex is shown in brackets. CP-115,953, and CP-115,955(163, The quinolones, CP-67,804, 176),as well as genistein' and the nitroimidazole Ro 15-0216 (162,246) appear to act primarily by stimulating the rate of enzyme-mediated DNA cleavage. In contrast, etoposide (118, appear to act primarily by 247) and amsacrine (118,246,248) inhibiting the rate of DNA religation.
by impairing the ability of the enzyme to rejoin breaks made in the backbone of DNA. Other drugs, such as the quinolone CP-115,953 [which is an even more potent stimulator of DNA breakage than is etoposide (163,178)1, have little effect on rates of topoisomerase II-mediated DNA religation (162,163,176,246).4 Although it has yet to be rigorously demonstrated, this latter finding strongly suggests that the second mechanistic class of drugs acts primarily by increasing the forward rate of enzymemediated DNA scission. Mechanistic differences between drug classes do not correlate with the mode of drug-DNA binding. For example, while etoposide and amsacrine both inhibit DNA religation, the former is nonintercalative in nature (147, 149) while the latter is strongly intercalative (136-138). Furthermore, the quinolone CP-115,953,whichshows little ability to inhibit DNA religation, is nonintercalative like etoposide (163). Thus, the historical characterization of topoisomerase II-targeted agents simply on the basis of their DNA binding properties no longer appears to be an appropriate means for classifying their mechanism of action. Antineoplastic agents enhance topoisomerase II-mediated DNA breakage in the presence or absence of ATP or in the presence of nonhydrolyzable ATP analogs that support the DNA strand passage event of the enzyme but not turnover (71-73,118). Therefore, the topoisomerase II-DNA cleavage complexes established both prior to and following DNA strand passage appear to be drug targets. In all cases that have been reported, the mechanism by which any given compound enhanced DNA breakage before and after strand passage (i.e., inhibition of religation us stimulation of DNA cleavage) was identical (118,163h4 Finally, in addition to their effects on the DNA cleavage/ religation equilibrium of topoisomerase 11,some (but not all) antineoplastic agents affect other steps of the enzyme's catalytic cycle. For example, amsacrine, genistein, and CP-115,953impair the ability of topoisomerase I1 to carry out its DNA strand passage event (249) and to hydrolyze its ATP cofactor (250). In contrast, etoposide has little effect on either of these critical reaction steps (249,250). These findings undercut the common assumption that all antineoplastic agents are specific for the DNA scission/ reunion reaction mediated by the enzyme. I t is not yet 4M. J. Robinson, A. H. Corbett, and N. Osheroff, unpublished observations.
Corbett and Osheroff
clear whether any of these inhibitory activities contribute to the cytotoxic nature of topoisomerase II-targeted drugs. However, as described in the followingsection, mechanistic differences between drug classes can be exploited to define relationships between drug interaction domains on topoisomerase 11.
Drug Interaction Domains on Topoisomerase I1 The only information concerning amino acid residues that are important for interactions between topoisomerase I1 and antineoplastic agents comes from the characterization of mutant drug-resistant enzymes. Amino acid alterations identified to date in type I1 topoisomerases from resistant cell lines are shown in Table 11. All of the enzymes listed in this table display resistance to antineoplastic agents in vitro (213-223). Thus far, mutations cluster in two regions in topoisomerase 11. One is located in the gyrB homology domain of the enzyme and spans one of the two consensus ATP recognition motifs (residues 472-477) in topoisomerase I1 (77,224,226,227). The other is located in the gyrA domain of the enzyme and spans the active site tyrosine (residue 805) involved in DNA cleavage (221,222,225,228).The fact that many mutations associated with drug resistance in the eukaryotic type I1 enzyme are found in the gyrB homology domain is surprising considering that most clinically relevant mutations in gyrase that confer high levels of resistance to quinolone-based antimicrobials map to the A subunit of the prokaryotic enzyme (75,251-254). While genetic studies have provided important information regarding the interaction of topoisomerase 11with antineoplastic agents and have defined mutations that confer drug resistance upon the eukaryotic enzyme, they have not been able to define relationships between the interaction domains for specific drug classes. Generalizations concerning drug interaction domains have been confounded by the fact that many mutant type I1 enzymes display distinctly different and often contradictory profiles of drug resistance. For example, while the CEM/VM-1 and CEM/VM-1-5 enzymes display resistance to all classes of DNA cleavage-enhancing drugs (216,217),the HL60/ AMSA and KBM-3/AMSA enzymes show high resistance only to intercalative agents (219, 220). Moreover, the VpmR-5enzyme displays a broad drug resistance profile but is highly sensitive to quinolone-based compounds (163, 218Ia3Thus, on the basis of genetic evidence alone, it is not clear which (if any) DNA cleavage-enhancing drugs share a common interaction domain on the type I1 enzyme. Recently, a biochemical approach that defines relationships between drug interaction domains on topoisomerase I1 has been developed (249,250). Although this technique does not identify amino acid residues that are involved in enzyme-drugbinding, it can readily distinguish whether two compounds interact with topoisomerase I1 at sites that overlap or are distinct from one another. Thus, the biochemical and genetic approaches to defining enzymedrug interactions complement one another. The biochemical approach takes advantage of the finding that many antineoplastic compounds have different effects on the various steps of the topoisomerase I1 catalytic cycle. Mechanistic differences are exploited to design a series of competition experiments that categorize drug interaction domains on the enzyme (249,250). While studies employing this approach have been limited in scope, results indicate that the interaction domain on
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 691
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Table 11. Drug Resistance Mutations in Topoisomerase 11 enzymea
CEM/VM-1 CEM/VM-1-5
HL-GO/AMSA KBM-3lAMSA VPMR-5 CEM/VP-1 top2-5
species human human human human Chinese hamster human yeast
drug selection teniposide teniposide
I
mutation*
homology domain
RW-CBC
s
gyrB gyrA
224
K
gyrB
77,226
gyrB gyrA gyrA gyrA gyrA gyrA gyrA
227 228 221
p803 -.
t
Re7
amsacrine teniposide ' etoposide etoposide/amsacrine
R4ru K7ee
-.Q +
+
N
RSOSP '
k--I top2-101 top2-103
Mw-1 yeast yeast
G7so
etoposide/amsacrine etoposide/amsacrine
P-
+
D
sc
-.
refs
225
222 222
*
a All of the enzymes listed display drug resistance in vitro. Sequence positions correspond to the sequence of human topoisomerase I1 (76) as amended by Hinds et al. (77)to facilitate direct comparisons. It should be noted that the amended sequence contains one additional amino acid in the region of residues 109-114. The sequence positions of mutated residues listed in this table reflect this correction in the primary
structure of the enzyme. 0 These mutations have been shown to confer drug resistance by site-directed mutagenesis (222).6
topoisomerase I1 for etoposide overlaps those of several other DNA cleavage-enhancingdrugs including amsacrine, genistein, and CP-115,953 (249). Although there is an obvious need for additional studies, these initial findings suggest that most if not all antineoplastic agents share a common interaction domain on the eukaryotic enzyme.
Summary and Perspectives Like the elixir that transformed the respectable Dr. Jekyll into the murderous Mr. Hyde (255),drugs targeted to topoisomerase I1 convert this essential enzyme into a potent physiological toxin. This transmogrification subsequently triggers a cascade of events that induces treated cells to commit ritualistic suicide (205, 207, 208). Considering the clinical importance of topoisomerase 11-targeted drugs, a number of issues concerning their mechanism of action demand further attention. First, the cellular processing of drug-stabilized enzymeDNA cleavage complexes needs to be characterized. For example, by manipulating the pathways involved in the repair of enzyme-mediated DNA breaks, it may be possible to enhance the lethality of topoisomerase 11-targetedagents. This has already been accomplished in simple laboratory models (196, 256, 257). Second, the impact of drug mechanism on cytotoxicity has yet to be fully explored. One important question is whether agents that enhance nucleic acid breakage by inhibiting the rate of topoisomerase 11-mediated DNA religation have an inherent cytotoxic advantage over drugs that act by stimulating rates of DNA cleavage. Initial studies indicate that this may be the case. While the quinolone CP-115,953 (which acts by the latter mechanism) is considerably more potent than etoposide (which acts by the former mechanism) in vitro, the two drugs are equipotent in vivo (163,178).Third, interactions between antineoplastic agents and topoisomerase I1 need to be described in considerably greater detail. As of this writing, no topoisomerase IIQdrugbinding studies have been reported. Furthermore, it is not known whether the amino acids identified by genetic studies form specific contacts with antineoplastic agents or whether amino acid substitutions that confer drug resistance act by inducing global changes in the enzyme. Ultimately, crystallization of enzymedrug complexes will be required to resolve this critical point. In the decade that has passed since topoisomerase I1 was first identified as a target for antineoplastic agents, tremendous strides have been made in our understanding SJ.
Nitiss, personal communication.
of how these drugs function. However, it is obvious that our knowledge is still rudimentary. Hopefully, as our analysis of drug action becomes more sophisticated, we will be in a better position to fully exploit the dualistic nature of topoisomerase I1 (i.e., essential enzyme/cellular toxin) and develop more effective therapies against human cancers.
Acknowledgment. This work was supported by National Institutes of Health Grants GM33944 and DK43325 and by American Cancer Society Research Grant NP-812 and Faculty Research Award FRA-370. We are grateful to Edna Kunkel for expertise in graphic design, Jackie Rule for expert photography, Milind Karkare for assistance with computer formatting, Susan Heaver for conscientious preparation of the manuscript, and Sarah Elsea and Stacie Jo Froelich-Ammon for critical reading of the manuscript. References Wang, J. C. (1985) DNA topoisomerases. Annu. Rev.Biochem. 54, 665-697.
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