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
---
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
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 587
Invited Reviews
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
Invited Reviews
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.
Osheroff, N. (1989) Biochemical basis for the interactions of type I and type I1 topoisomerases with DNA. Pharmacol. Ther. 41,223241.
Champoux,J.J. (1990) Mechanisticaspectaoftype-Itopohmerasee. In DNA Topology and Its Biological Effects (Cozzarelli, N. R., and Wang, J. C., Eds.) pp 217-242, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Hsieh, T. (1990) Mechanistic aspectaof type-IIDNAtopoisomerasee. In DNA Topology and Its Biological Effects (Cozzarelli, N. R.,and Wang, J. C., Eds.) pp 242-263, Cold Spring Harbor Laboratory Prees, Cold Spring Harbor, NY. Liu, L., and Miller, K. G. (1981) Eukaryotic DNA topoisomerases: Two forms of type I topoisomerases from HeLa cell nuclei. h o c . Natl. Acad. Sei. U.S.A. 78, 3487-3491. Tricoli,J. V., and Kowalski,D. (1983) Topoisomerase I from chicken erythrocytes: Purification, characterization, and detection by a deoxyribonucleic acid binding assay. Biochemistry 22,2025-2031. Goto, T., Laipis, P., and Wang, J. C. (1984) The purification and characterization of DNA topoisomerases I and I1 of the yeast Saccharomyces cerevisiae. J.Biol. Chem. 259, 10422-10429. Pulleyblank, D. E., Shure, M., Tang, D., Vinogad, J., and Vosberg, H. (1975) Action of nicking-closing enzyme on supercoiled and nonsupercoiled closed circular DNA Formation of a Boltzman distribution of topological isomers. R o c . Natl. &ad. Sci. U.S.A. 72,4280-4284.
Champoux, J. J. (1976) Evidence for an intermediate with a singlestrand break in the reaction catalyzed by the DNA untwisting enzyme. Roc. Natl. Acad. Sci. U.S.A. 73, 3488-3491. Miller, K. G., Liu, L. F., and Englund, P. T. (1981) A homogeneous type I1 DNA topoisomerase from HeLa cell nuclei. J.Biol. Chem. 256,9334-9339.
Sander, M., and Hsieh, T.4. (1983) Double-strand DNA cleavage by type I1 DNA topoisomerase from Drosophila melanogaster. J. Biol. Chem. 258,8421-8428. Shelton, E. R., Osheroff, N., and Brutlag, D. L. (1983) DNA topoisomerase I1 from Drosophila melanogaster: Purification and physical characterization. J. Biol. Chem. 258, 9530-9535.
592 Chem. Res. Toxicol., Vol. 6, No. 5, 1993 (13) Halligan, B. D., Edwards, K. A., and Liu, L. F. (1985) Purification and characterization of a type I1 DNA from bovine calf thymus. J. Biol. Chem. 260, 2475-2482. (14) Schomburg, U., and Grosse, F. (1986) Purification and characterization of DNA topoisomerase I1 from calf thymus associated with polypeptides of 175 and 150 kDa. Eur. J.Biochem. 160,451-457. (15) Osheroff, N., Shelton, E. R., and Brutlag, D. L. (1983) DNA topoisomerase I1 from Drosophila melanogaster: Relaxation of supercoiled DNA. J. Biol. Chem. 258,9536-9543. (16) Hsieh, T.-S., and Brutlag, D. (1980) ATP-dependent DNA topoisomerase from D. melanogaster reversibly catenates duplex DNA rings. Cell 21, 115-121. (17) Liu, L. F., Liu, C.-C., and Alberta, B. M. (1980) Type I1 DNA topoisomerases: Enzymes that can unknot a topologically knotted DNA molecule uia a reversible double-strand break. Cell 19,697707. (18) Thrash, C., Voelkel, K., DiNardo, S., and Sternglanz, R. (1984) Identification of Saccharomycescereukiae mutants deficientin DNA topoisomerase I activity. J.Biol. Chem. 259, 1375-1377. (19) Uemura, T., and Yanagida, M. (1984) Isolation of type I and type I1 topoisomerase mutants from fission yeast: Single and double mutants show different phenotypes in cell growth and chromatin organization. EMBO J. 3, 1737-1744. (20) Goto, T., and Wang, J. C. (1985) Cloning of yeast TOPI, the gene encoding DNA topoisomerase I, and construction of mutants defective in both DNA topoisomerase I and DNA topoisomerase 11. Roc. Natl. Acad. Sci. U.S.A. 82, 7178-7182. (21) Hsieh, T., Lee, M., Crenshaw, D., Brown, S., and Chen, A. (1993) Structuresand functions of DNAtopoisomerases. J.Cell. Biochem. Suppl. 17C, 150. (22) Fleischmann, G., Pflugfelder, G., Steiner, E. K., Javaherian, K., Howard, G. C., Wang, J. C., and Elgin, S. C. R. (1984) Drosophila DNA topoisomerase I is associated with transcriptionally active regions of the genome. Roc. Natl. Acad. Sci. U.S.A. 81,6958-6962. (23) Muller, M. T., Pfund, W. P., Mehta, V. B., and Trask, D. K. (1985) Eukaryotic type I topoisomerase is enriched in the nucleolus and catalytically active on ribosomal DNA. EMBO J.4, 1237-1243. (24) Gilmour, D. S., Pflugfelder, G., Wang, J. C., and Lis, J. T. (1986) Topoisomerase I interacts with transcribed regions in Drosophila cells. Cell 44, 401-407. (25) Egyhazi, E., and Durban, E. (1987) Microinjection of anti-topoisomerase I immunoglobulin G into nuclei of Chironomw tentans salivary gland cells leads to blockage of transcription elongation. Mol. Cell. Biol. 7, 4308-4316. (26) Zhang, H., Wang, J. C., and Liu, L. F. (1988) Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes. Proc. Natl. Acad. Sci. U S A . 85, 1060-1064. (27) Kroeger, P. D., and Rowe, T. C. (1992) Analysis of topoisomerase I and I1cleavage sites on the Drosophila actin and Hap70 heat shock genes. Biochemistry 31, 2492-2501. (28) Champoux, J. J. (1988) Topoisomerase I is preferentially associated with isolated replicating simian virus 40 molecules after treatment of infected cella with camptothecin. J. Virol. 62, 3675-3683. (29) Kim, R. A., and Wang, J. C. (1989) Function of DNAtopoisomerases as replication swivels in Saccharomyces cereuisiae. J.Mol. Biol. 208,257-267. (30) Wang, J. C., and Liu, L. F. (1990) DNA Replication: Topological aspects and the roles of DNA topoisomerases. In DNA Topology and Its Biological Effects (Cozzarelli, N. R., and Wang, J. C., Eds.) pp 321-340, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (31) Snapka, R. M., and Permana, P. A. (1993) SV40 DNA replication intermediates: Analysis of drugs which target mammalian DNA replication. BioEssays 15, 121-127. (32) Higashinakagawa,T., Wahn, H., and Reeder, R. H. (1977) Isolation of ribosomal gene chromatin. Deu. Biol. 55, 375-386. (33) Weisbrod, S. T. (1982) Properties of active nucleosomes as revealed by HMG 14 and 17 chromatography. Nucleic Acids Res. 10,20172042. (34) Bodley, A. L., Wu, H.-Y., and Lui, L. F. (1987) Regulation of DNA topoisomerases during cellular differentiation. Natl. Cancer Inst. Monogr. 4, 31-35. (35) Heck, M. M. S.,Hittelman, W. N., and Earnshaw, W. C. (1988) Differential expression of DNA topoisomerases I and I1 during the eukaryotic cell cycle. Roc. Natl. Acad. Sci. U.S.A. 85,1086-1090. (36) Hsiang, Y.-H., Wu, H.-Y., and Liu, L. F. (1988) Proliferationdependent regulation of DNA topoisomerase I1 in cultured human cells. Cancer Res. 48,3230-3235. (37) DiNardo, S., Voelkel, K., and Sternglanz, R. (1984) DNA topoisomerase I1 mutant of Saccharomyces cereuisiae: Topoisomerase I1is required for segregationof daughter moleculea at thetermination of DNA replication. Proc. Natl. Acad. Sci. U.S.A. 81, 26162620. (38) Goto, T., and Wang, J. C. (1984) Yeast DNA topoisomerase I1 is encoded by a single copy, essential gene. Cell 36, 1073-1080.
Corbett and Osheroff (39) Holm, C., Goto,T., Wang, J. C., and Botstein, D. (1985) DNA topoisomerase I1 is required at the time of mitosis in yeast. Cell 41, 553-563. (40) Uemura, T., and Yanagida, M. (1986) Mitotic spindle pulla but fails to separate chromosomes in type I1 DNA topoisomerase mutants: Uncoordinated mitosis. EMBO J. 5, 1003-1010. (41) Uemura, T., Ohkura, H., Adachi, Y., Morino, K., Shiozaki, K., and Yanagida, M. (1987) DNA topoisomerase I1 is required for condensation and separation of mitotic chromosomesin S.pombe. Cell 60,917-925. (42) Yang, L., Wold, M. S., Li, J. J., Kelly, T. J., and Liu, L. F. (1987) Roles of DNA topoisomerases in simian virus 40 DNA replication in vitro. R o c . Natl. Acad. Sci. U.S.A. 84, 950-954. (43) Ishimi,Y., Sugaaawa,K.,Hanaoka, F.,Eki,T.,andHurwitz, J. (1992) Topoisomerase I1 plays an essential role as a swivelase in the late stage of SV40 chromosome replication in vitro. J.Biol. Chem. 267, 462-466. (44) Bee, Y.-S., Kawasaki, I., Ikeda, H., and Liu, L. F. (1988) Illegitimate recombination mediated by calf thymus DNA topoisomerase I1 in vitro. Proc. Natl. Acad. Sci. U.S.A. 85, 2076-2080. (45) Dillehay, L. E., Jacobson-Kram, D., and Williams, J. (1989) DNA topoisomerases and models of sister chromatid exchange. Mutat. Res. 215, 15-23. (46) Kim, R. A., and Wang, J. C. (1989) A subthreshold level of DNA topoisomerases leads to the excision of yeast rDNA as extrachromosomal rings. Cell 57, 975-985. (47) Ikeda, H. (1990) DNA topoisomerase-mediated illegitimate recombination. In DNA Topology and Its Biological Effects (Cozzarelli, N. R., and Wang, J. C., Eds.) pp 341-359, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (48) Rose, D., Thomas, W., and Holm, C. (1990) Segregationof recombined chromosomes in meiosis I requires DNA topoisomerase 11. Cell 60, 1009-1017. (49) Wang, J. C., Caron, P. R., and Kim, R. A. (1990) The role of DNA topoisomerases in recombination and genome instability: A double edged sword? Cell 62,403-406. (50) Earnshaw, W. C., Halligan, B., Cooke, C. A., Heck, M. M. S.,and Liu, L. F. (1985) Topoisomerase I1 is a structural component of mitotic chromosome scaffolds. J. Cell Biol. 100, 1706-1715. (51) Eamshaw, W. C., and Heck, M. M. S. (1985) Localization of topoisomerase I1 in mitotic chromosomes. J.Cell Biol. 100, 17161725. (52) Gasser, S. M., and Laemmli, U. K. (1986) The organisation of chromatin loops: Characterization of a scaffold attachment site. EMBO J. 5,511-518. (53) Gasser, S. M., LaRoche, T., Falquet, J., Boy de la Tour, E., and Laemmli, U. K. (1986) Metaphase chromosome structure: Involvement of topoisomerase 11. J. Mol. Biol. 188, 613-629. (54) Newport,J. (1987) Nuclear reconstitution invitro: Stages of assembly around protein-free DNA. Cell 48, 205-217. (55) Newport, J., and Spann, T. (1987) Disassembly of the nucleus in mitotic extracte; membrane vesicularization,lamin disassembly,and chromosome condensation are independent processes. Cell 48,219230. (56) Wood, E. R., and Earnshaw, W. C. (1990) Mitotic chromatin condensation in vitro using somatic cell extracts and nuclei with variable levels of endogenous topoisomerase 11. J. Cell Biol. 111, 2839-2850. (57) Adachi, Y., Luke, M., and Laemmli, U. K. (1991) Chromosome assembly in vitro: Topoisomerase I1 is required for condensation. Cell 64, 137-148. (58) Hirano, T., and Mitchison, T. J. (1991) Cell cycle control of higherorder chromatin assembly around naked DNA in vitro. J.Cell Biol. 115, 1479-1489. (59) Hirano, T., and Mitchison, T. J. (1993) Topoisomerase I1 does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopw egg extracts. J. Cell Biol. 120, 601-612. (60) Shamu, C. E., and Murray, A. W. (1992) Sister chromatid separation in frog extracts requires DNA topoisomerase I1 activity during anaphase. J. Cell Biol. 117, 921-934. (61) Duget, M., Lavenot, C., Harper, F., Mirambeau, G., and De Recondo, A. (1983) DNA topoisomerases from rat liver: Physiological variations. Nucleic Acids Res. 11, 1059-1075. (62) Heck, M. M. S., and Eamshaw, W. E. (1986) Topoisomerase 11: A specific marker for cell proliferation. J. Cell Biol. 103,2569-2581. (63) Nelson, W., Cho, K., Hsiang, Y.-H., Liu, L. F., and Coffey, D. S. (1987) Growth-related elevations of DNA topoisomerase I1 levels found in Dunning R3327 rat prostatic adenocarcinomas. Cancer Res. 47, 32463250. (64)Sullivan, D. M., Latham, M. D., and Ross, W. E. (1987) Proliiferationdependent topoisomerase I1 content as a determinant of antineoplastic drug action in human, mouse, and Chinese hamster ovary cells. Cancer Res. 47,3973-3979.
Invited Reviews (65) Holden, J. A., Rolfson, D. H., and Wittwer, C. T. (1990) Human DNA topoisomerase I1 Evaluation of enzyme activity in normal and neoplastic tissues. Biochemistry 29, 2127-2134. (66) Berrios, M., Osheroff,N., and Fisher, P. A. (1985)Insitu localization of DNA topoisomerase 11, a major polypeptide component of the Drosophila nuclear matrix. Proc. Natl. Acad. Sci. U.S.A. 82,41424146. (67) Chow, K.-C., and Ross, W. E. (1987)Topoisomerase 11-specificdrug sensitivity in relation to cell cycle progression. Mol. Cell Biol. 7, 3119-3123. (68) Champoux, J. J., and Dulbecco, R. (1972) An activity from mammalian cells that untwists superhelical DNA-a possible swivel for DNA replication. Proc. Natl. Acad. Sci. U.S.A. 69, 143-146. (69) Champoux, J. J., and McConaughy, B. L. (1976) Purification and characterization of the DNA untwisting enzyme from rat liver. Biochemistry 16, 4638-4642. (70) Baldi, M. I., Beedetti, P., Mattocia, E., and Tocchini-Valentini, G. P. (1980) In vitro catenation and decatenation of DNA and a novel eukaryotic ATP-dependent topoisomerase 11. Cell 20,461-467. (71) Liu, L. F. (1989)DNAtopobomerasespobonsasantineoplastic drugs. Annu. Reu. Biochem. 68,351-375. (72) Liu, L. F. (1990)Anticancer drugs that convert DNAtopoisomerases into DNA damaging agents. In DNA Topology and Its Biological Effects (Cozzarelli,N. R., and Wang, J. C., Eds.) pp 371-389, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (73) Schneider, E., Hsiang, Y.-H., and Liu, L. F. (1990) DNA topoisomerases as anticancer drug targets. Adu. Pharmacol. 21, 149183. (74) Pommier, Y. (1993) DNA topoisomerase I and I1 in cancer chemotherapy: Update and perspectives. Cancer Chemother. Pharmacol. 32, 103-108. (75) Reece, R. J., and Maxwell, A. (1991) DNA gyrase: Structure and function. CRC Crit. Reu. Biochem. Mol. Biol. 26, 335-375. (76) Tsai-Pflugfelder, M., Liu, L. F., Liu, A. A,, Tewey, K. M., WhangPeng, J., Knutaen, T., Huebner, K., Croce, C. M., and Wang, J. C. (1988) Cloning and sequencing of cDNA encoding human DNA topoisomerase I1 and localization of the gene to chromosomeregion 17q21-22. Proc. Natl. Acad. Sei. USA. 85, 7177-7181. (77) Hinds, M., Deisseroth, K., Mayes, J., Altschuler, E., Jansen, R., Ledley, F. D., and Zwelling, L. A. (1991) Identification of a point mutation in the topoisomerase I1 gene from a human leukemia cell line containing an amsacrine-resistant form of topoisomerase 11. Cancer Res. 51,4729-4731. (78) Lynn,R.,Giaever,G., Swanberg,& L.,and Wang,J. C. (1986)Tandem regions of yeast DNA topoisomeraseI1share homology with different subunits of bacterial gyrase. Science 233,647-649. (79) Wyckoff, E., Natalie, D., Nolan, J. M., Lee, M., and Hsieh, T.-S. (1989) Structure of the Drosophila DNA topoisomerase I1 gene: Nucleotide sequence and homology among topoisomerases 11. J. Mol. Biol. 205, 1-13. (80) Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) Distantly related sequences in the a- and &subunits of ATP synthetase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide folding domain. EMBO J. 1, 945-951. (81) Saraste,M., Sibbald,P. R., and Wittinghofer, A. (1990)TheP-loop-a common motif in ATP- and GTP-binding proteins. Trends Biol. Sci. 15, 430-434. (82) Worland, S. T., and Wang, J. C. (1989) Inducible overexpression, purification, and active site mapping of DNA topoisomerase I1 from the yeast Saccharomyces cereuisiae. J.Biol. Chem. 264,4412-4416. (83) Cardenas, M. E., Dang, Q., Glover, C. V. C., and Gasser, S.M. (1992) Casein kinase I1 phosphorylates the eukaryote-specific C-terminal domain of topoisomerase I1 in vivo. EMBO J. 11, 1785-1796. (84) Corbett, A. H., Fernald, A. W., and Osheroff, N. (1993) Protein kinase C modulates the catalytic activity of topoisomerase I1 by enhancing the rate of ATP hydrolysis: Evidence for a common mechanism of regulation by phosphorylation. Biochemistry 32, 2090-2097. (85) Giaever, G.,Lynn, R., Goto,T., and Wang, J. C. (1986)The complete nucleotide sequence of the structural gene TOP2 of yeast DNA topoisomerase 11. J. Biol. Chem. 261, 12448-12454. (86) Voelkel-Meiman, K., DiNardo, S., and Sternglanz, R. (1986) Molecular cloning and genetic mapping of the DNA topoisomerase I1 gene of Saccharomyces cereuisiae. Gene 42, 193-199. (87) Uemura, T., Morikawa, K., and Yanagida, M. (1986)The nucleotide sequence of the fission yeast DNA topoisomerase I1gene: Structural and functional relationships to other DNA topoisomerases. EMBO J. 5, 2355-2361. (88) Nolan, J.M.,Lee,M.P.,Wyckoff,E.,andHsieh,T.-S. (1986)Isolation and characterization of the gene encoding Drosophila DNA topoisomerase 11. Proc. Natl. Acad. Sei. U.S.A. 83,3664-3668. (89) Drake, F. H., Zimmerman, J. P., McCabe, F. L., Bartus, H. F., Per, S. R., Sullivan, D. M., Ross, W. E., Mattern, M. R., Johnson, R. K., Crooke, S. T., and Mirabelli, C. K. (1987) Purification of topoi-
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 593 somerase I1from amsacrine-resistant P388 leukemia cells Evidence for two forms of the enzyme. J. Biol. Chem. 262,16739-16747. (90) Chung, T. D. Y., Drake, F. H., Tan, K. B., Per, S. R., Crooke, S.T., and Mirabelli, C. K. (1989) Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase I1 isozymes. Proc. Natl. Acad. Sci. USA. 86,9431-9435. (91) Austin, C. A., Sng, J. H., Patel, S., and Fisher, L. M. (1993) Novel HeLa topoisomerase I1 is the 11-beta isoform-Complete coding sequence and homologywith other type I1topoisomerases. Biochim. Biophys. Acta 1172, 283-291. (92) Austin, C. A., and Fisher, L. M. (1990)Isolation and characterization of a human cDNA clone encoding a novel DNA topoisomerase I1 homologue from HeLa cells. FEBS Lett. 266, 115-117. (93) Jenkins, J. R., Ayton, P., Jones, T., Davies, S. L., Simmons, D. L., Harris, A. L., Sheer, D., and Hickson, I. D. (1992) Isolation of cDNA clones encoding the @ isozyme of human DNA topoisomerase I1 and localization of the gene to chromosome 3p24. Nucleic Acids Res. 20,5587-5592. (94) Tan, K. B., Dorman, T. E., Falls, K. M., Chung, T. D. Y., Mirabelli, C. K., Crooke, S. T., and Mao, J. (1992) Topoisomerase IIa and topoisomerase IIB genes: Characterization and mapping to human chromosomes 17 and 3, respectively. Cancer Res. 52, 231-234. (95) Osheroff, N., Zechiedrich, E. L., and Gale, K. C. (1991) Catalytic function of DNA topoisomerase 11. BioEssays 13, 269-283. (96) Drake, F. H., Hofman, G. A., Bartus, H. F., Mattern, M. R., Crooke, S. T., and Mirabelli, C. K. (1989) Biochemical and pharmacological propertiesof p170andp180 forms of topoisomeraseI1. Biochemiatry 28,8154-8160. (97) Woessner, R. D., Chung, T. D. Y., Hofman, G. A,, Mattern, M. R., Mirabelli, C. K., Drake, F. H., and Johnson, R. K. (1990)Differences between normal and ras-transformed NIH-3T3 cells in expression of the 170kD and the 180kD forms of topoisomerase 11. Cancer Res. 50, 2901-2908. (98) Woessner, R. D., Mattern, M. R., Mirabelli, C. K., Johnson, R. K., and Drake, F. H. (1991) Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase I1 in NIH-3T3 cells. Cell Growth Differ. 2, 209-214. (99) Holden, J. A., Rolfson, D. H., and Wittwer, C. T. (1992) The distribution of immunoreactive topoisomerase I1 protein in human tissues and neoplasms. Oncol. Res. 4, 157-166. (100) Capranico, G., Tinelli, S., Austin, C. A., Fisher, M. L., and Zunino, F. (1992) Different patterns of gene expression of topoisomerase I1 isoforms in differentiated tissues during murine development. Biochim. Biophys. Acta 1132,43-48. (101) Zini, N., Martelli, A. M., Sabatelli, P., Santi, S.,Negri, C., Astaldi Ricotti, G. C. B., Maraldi, N. M. (1992) The 180-kDa isoform of topoisomerase I1 is localized in the nucleolus and belongs to the structural elements of the nucleolar remnant. Exp. Cell Res. 200, 460-466. (102) Lindsley, J. E., and Wang, J. C. (1991) Proteolysis patterns of epitopically labeled yeast DNA topoisomerase I1 suggest an allosteric transition in the enzyme induced by ATP binding. Proc. Natl. Acad. Sci. U.S.A. 88,10485-10489. (103) Zechiedrich,E.L.,andOsheroff, N. (1990)Eukaryotictopoisomerase I1 recognizes nucleic acid topology by preferentially interacting with DNA crossovers. EMBO J. 9,4555-4562. (104) Howard, M. T., Lee, M. P., Hsieh, T.-S., and Griffith, J. D. (1991) Drosophila topoisomerase 11-DNA interactions are affected by DNA structure. J. Mol. Biol. 217, 53-62. (105) Lee, M. P., Sander, M.,and Hsieh,T.-S. (1989)Nucleaseprotection by Drosophila DNA topoisomerase 11. Enzyme/DNA contacts at the strong topoisomerase I1 cleavage sites. J. Biol. Chem. 264, 21779-21787. (106) Thomsen, B., Bendixen, C., Lund, K., Andersen, A. H., Ssrensen, B.S.,and Westergaard, 0.(1990)Characterizationoftheinteraction between topoisomeraseI1and DNA by transcriptional footprinting. J.Mol. Biol. 216, 237-244. (107) Osheroff, N. (1987) Role of the divalent cation in topoisomerase I1 mediated reactions. Biochemistry 26, 6402-6406. (108) Sander, M., Hsieh, T.-S., Udvardy, A., and Schedl, P. (1987) Sequence dependence of Drosophila topoisomerase I1 in plasmid relaxation and DNA binding. J. Mol. Bid. 194, 219-229. (109) Liu, L. F., Rowe, T. C., Yang, L., Tewey, K. M., and Chen, G. L. (1983)Cleavage of DNA by mammalian DNA topoisomerase 11. J. Biol. Chem. 268, 15365-15370. (110) Sander, M., and Hsieh, T.3. (1985) Drosophila topoisomerase I1 double-strand DNA cleavage: Analysisof DNA sequence homology at the cleavage site. Nucleic Acid Res. 13, 1057-1072. (111) Muller,M. T., Spitzner, J. R.,DiDonato, J. A.,Mehta, V. B.,Tsutsui, K., and Tsutsui, K. (1988) Single-strand DNA cleavages by eukaryotic topoisomerase 11. Biochemistry 27, 8369-8379. (112) Andersen, A. H., Christiansen, K., Zechiedrich, E. L., Jensen, P. S.,Osheroff, N., and Westergaard, 0. (1989) Strand specificity of
594 Chem. Res. Toxicol., Vol. 6, No. 5, 1993 the topoisomerase 11-mediated double-stranded DNA cleavage reaction. Biochemistry 28, 6237-6244. (113) Lund, K., Andersen, A. H., Christiansen, K., Svejstrup, J. Q., and Westergaard,0. (1990)MinimalDNArequirementof topoisomerase 11-mediatedcleavage in vitro. J. Biol. Chem. 265, 1385613863. (114) Rowe, T. C., Tewey, K. M., and Liu, L. F. (1984) Identification of the breakage-reunion subunit of T4 DNA topoisomerase. J.Biol. Chem. 259, 9177-9181. (115) Osheroff, N.,and Zechiedrich,E. L. (1987)Calcium-promotedDNA cleavage by eukaryotic topoisomerase 11: Trapping the covalent enzymeDNA complex in an active form. Biochemistry 26,43034309. (116) Osheroff, N. (1986)Eukaryotic topoisomerase 11: Characterization of enzyme turnover. J. Biol. Chem. 261,9944-9950. (117) Roca, J., and Wang, J. C. (1992)The capture of a DNAdouble helix by an ATP-dependent protein clamp: A key step in DNA transport by type I1 DNA topoisomerases. Cell 71,833-840. (118) Robinson, M. J., and Osheroff, N. (1991) Effects of antineoplastic drugs on the post-strand passage DNA cleavage/religation equilibrium of topoisomerase 11. Biochemistry 30, 1807-1813. (119) Lindsley, J. E., and Wang, 3. C. (1993) On the coupling between ATP usage and DNA transport by yeast DNA topoisomerase 11. J. Biol. Chem. 268,80968104. (120) Rottman,M.,Schrbder, H. C.,Gramzow,M.,Renneisen,K., Kurelec, B., Dorn, A,, Friese, U., and Mhller, W. E. G. (1987) Specific phosphorylation of proteins in pore complex-laminae from the sponge Geodia cydonium by the homologous aggregation factor and phorbol ester. Role of protein kinase C in the phosphorylation of DNA topoisomerase 11. EMBO J. 6, 3939-3944. (121) Ackerman, P., Glover, C. V. C., and Osheroff, N. (1988) Phosphorylation of DNA topoisomerase I1in vivo and in totalhomogenate8 of Drosophila Kc cells: The role of casein kinase 11. J.Biol. Chem. 263,12653-12660. (122) Heck, M. M. S., Hittelman, W. N., and Earnshaw, W. C. (1989) In vivo phosphorylation of the 170-kDa form of eukaryotic DNA topoisomerase 11 Cell cycle analysis. J.Biol. Chem. 264,1516115164. (123) Saijo, M., Enomoto, T., Hanaoka, F., and Ui, M. (1990)Purification and characterization of type I1 DNA topoisomerase from mouse FM3A cells: Phosphorylation of topoisomeraseI1and modification of ita activity. Biochemistry 29, 583-590. (124) Kroll, D. J., and Rowe, T. C. (1991) Phosphorylation of DNA topoisomerase I1 in a human tumor cell line. J. Biol. Chem. 266, 7957-7961. (125) Saijo, M., Ui, M., and Enomoto, T. (1992) Growth state and cell cycle dependent phosphorylation of DNA topoisomeraseI1in Swiss 3T3 cells. Biochemistry 31, 359-363. (126) Shiozaki, K., and Yanagida, M. (1992)Functional dissection of the phosphorylated termini of fission yeast DNA topoisomerase 11. J. Cell Biol. 119, 1023-1036. (127) Cardenas, M. E., and Gasser, S. M. (1993) Regulation of topoisomerase I1 by phosphorylation: A role for casein kinase 11. J.Cell Sci. 104, 219-225. (128) Constantinou, A., Henning-Chubb, C., and Huberman, E. (1989) Novobiocin- and phorbo1-12-myristate-13-acetate-induced differentiation of human leukemia cells associated with a reduction in topoisomerase I1 activity. Cancer Res. 49, 1110-1117. (129) Matthes, E., Langen, P., Brachwitz, H., Schrbder, H. C., Maidhof, A., Weiler, B. E., Renneisen, K., and Miiller, W. E. G. (1990) Alteration of DNA topoisomerase I1activity during infection of H9 cells by human immunodeficiency virus type 1 in vitro: A target for potential therapeutic agents. Antiviral Res. 13, 273-286. (130) Miiller, W. E. G., Uqarkovic, D., Gamulin, V., Weiler, B. E., and Schrbder, H. C. (1990) Intracellular signal transduction pathways in sponges. Electron Microsc. Rev. 3, 97-114. (131) Ackerman, P., Glover, C. V. C., and Osheroff, N. (1985) Phosphorylation of DNA topoisomerase I1 by casein kinase I 1 Modulation of eukaryotic topoisomerase I1 activity in vitro. Roc. Natl. Acad. Sci. USA. 82, 3164-3168. (132) Corbett, A. H., DeVore, R. F., and Osheroff, N. (1992) Effect of casein kinase &mediated phosphorylation on the catalytic cycle of topoisomerase I1 Regulation of enzyme activity by phosphorylation. J. Biol. Chem. 267, 20513-20518. (133) DeVore, R. F., Corbett, A. H., and Osheroff, N. (1992) Phosphorylation of topoisomerase I1 by casein kinase I1 and protein kinase C: Effects on enzyme-mediated DNA cleavage/religation and sensitivity to the antineoplastic drugs etoposide and 4’-(9-acridinylamino)methane-sulfon-m-anisidide. CancerRes. 62,2156-2161. (134) Cardenas, M. E., Walter, R., Hanna, D., and Gasser, S. M. (1993) Casein kinase I1 copurifies with yeast DNA topoisomerase I1 and re-activates the dephosphorylated enzyme. J. Cell Sci. 104,533543. (135) Sahyoun, N., Wolf, M., Besterman, J., Hsieh, T.-S., Sander, M., Levine, H., 111, Chang, K.-J., and Cuatrecaeas, P. (1986) Protein kinase C phosphorylates topoisomerase I1 Topoisomerase acti-
Corbett and Osheroff vation and its possible role in phorbol ester-induced differentiation of HL-60 cells. Biochemistry 83, 1603-1607. (136) Waring, M. J. (1981) DNA modifications and cancer. Annu. Rev. Biochem. 50, 159-192. (137) Wilson, W. R., Baguley, B. C., Wakelin, L. P. G., and Waring, M. J. (1981) Interaction of the antitumor drug 4’-(9-acridinylamino)methanesulfon-m-anisidideand related acridineswith nucleic acids. Mol. Pharmacol. 20,404-414. (138) Nelson, E. M., Tewey, K. M., and Liu, L. F. (1984) Mechanism of antitumor drug action: Poisoning of mammalian DNA topoisomerase I1 by 4’-(9-acridinylamino)methanesulfon-m-anisidide. Roc. Natl. Acad. Sei. U.S.A. 81, 1361-1365. Pinedo, H. M., Chabner, B. A., and Longo, D. L. (1988) Cancer Chemotherapy and BiologicalResponse Modifications, Annual 9, Elsevier, Amsterdam. Kapuscinski, J., and Darzynkiewicz, Z. (1985) Interactions of antitumor agents ametatrone and mitoxantrone (Novatrone) with double-stranded DNA. Biochem. Pharmacol. 34,4203-4213. Tewey, K. M., Chen, G. L., Nelson, E. M., and Liu, L. F. (1984) Intercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase 11. J. Biol. Chem. 269,9182-9187. van der Graaf, W. T., and de Vries, E. G. (1990) Mitoxantrone: Bluebeard for malignancies. Anticancer Drugs 1, 109-125. Faulds, D., Balfour, J. A., Chrisp, P., and Langtry, H. D. (1991) Mitoxantrone: A review of ita pharmacodynamic and pharmacokinetic properties, and therapeutic potential in the chemotherapy of cancer. Drugs 41,400-449. Tewey, K. M., Rowe, T. C., Yang, L., Halligan, B. D., and Liu, L. F. (1984) Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase 11. Science 226,466-468. Potmesil, M., and Silber, R. (1990)DNA topoisomerases in clinical oncology. In DNA Topology and its BiologicalEjfects (Cozzarelli, N. R., and Wang, J. C., Eds.) pp 391-407, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sinha, B. K., and Politi, P. M. (1990) Anthracyclines. Cancer Chemother. Biol. Response Mod. 11, 45-57. Chen, G. L., Yang, L., Rowe, T. C., Halligan, B. D., Tewey, K. M., and Liu, L. F. (1984) Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase 11. J. Biol. Chem. 269, 13560-13566. Chow, K.-C.,MacDonald,T.L.,andRoss, W. E. (1988)DNAbinding by epipodophyllotoxina and N-acyl anthracyclines: Implications for mechanism of topoisomerase I1 inhibition. Mol. Pharmacol. 34,467-473. Ross, W., Rowe, T., Glisson, B., Yalowich, J., and Liu, L. F. (1984) Role of topoisomerase I1in mediating epipodophyllotoxin-induced DNA cleavage. Cancer Res. 44, 5857-5860. Bishop,J. F. (1992)Etoposidein the treatmentof leukemias.Semin. Oncol. 19, 33-38. Carney, D. N., Keane, M., and Grogan, L. (1992) Oral etoposide in small cell lung cancer. Semin. Oncol. 19,4044. DeVore, R., Hainsworth, J., Breco, F. A., Hande, K., and Johnson, D. (1992) Chronic oral etoposide in the treatment of lung cancer. Semin. Oncol. 19, 28-35. Nichols, C. R. (1992) The role of etoposide therapy in germ cell cancer. Semin. Oncol. 19, 72-77. Hsiang, Y.-H., Jiang, J. B., and Liu, L. F. (1989) Topoisomerase 11-mediatedDNA cleavageby a m o d i d e and its structural analogs. Mol. Pharmacol. 36, 371-376. Malviya, V. K., Liu, P. Y., Alberts, D. S., Suwit, E. A., Craig, J. B., and Hannigan, E. V. (1992)Evaluation of amondide in cervical cancer, phase 11. A SWOG study. Am. J.Clin. Oncol. 15,41-44. Perez, R. P., Nash, S. L., Ozols, R. F., Comis, R. L., and ODwyer, P. J. (1992)Phase I1studiesof a m o d i d e in advanced and recurrent sarcoma patients. Znuest. New Drugs 10, 99-101. Buzdar, A. U., Hortobagyi, G. N., Esparza, L. T., Holmes, F. A,, Ro, J. S., Fraschini, G., and Lichtiger,B. (1990)Elliptiniumacetate in metastatic breast cancer-a phase I1 study. Oncology 47,101104. Kayitalire, L., Thomas, F., Le Chevalier, T., Toussaint, C., Tursz, T., and Spielmann, M. (1992) Phase I1 study of a combination of elliptinium and vinblastine in metastatic breast cancer. Inuest. New Drugs 10, 303-307. Markovits, J., Linassier, C., Fosae, P., Couprie, J., Pierre, J., Jacquemin-Sablon, A., Saucier, J.-M., Le Pecq, J.-B., and Larsen, A. K. (1989) Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase 11. Cancer Res. 49, 5111-5117. Yamashita, Y., Kawada, S., and Nakano, H. (1990) Induction of mammalian topoisomerase I1 dependent DNA cleavage by nonintercalativeflavonoids,genistein and orobol. Biochem.Phrrrmacol. 39,737-744. Austin, C. A., Patel, S., Ono, K., Nakane, H., and Fisher, L. M. (1992) Site-specific DNA cleavage by mammalian DNA topoi-
Invited Reviews somerase I1 induced by novel flavone and catechin derivatives. Biochem. J. 282, 883-889. (162) Sorensen, B. S., Jensen, P. S.,Andersen, A. H., Christiansen, K., Alsner, J., Thomsen, B., and Westergaard, 0. (1990). Stimulation of topoisomerase I1mediated DNA cleavage by the 2-nitroimidazole Ro 15-0216. Biochemistry 29,9507-9515. (163) Robinson, M. J., Martin, B. A., Gootz, T. D., McGuirk, P. R., Moynihan, M., Sutcliffe, J. A., and Osheroff, N. (1991) Effects of quinolone derivatives on eukaryotic topoisomerase 11: A novel mechanism for enhancement of enzyme-mediated DNA cleavage. J. Biol. Chem. 266, 14585-14592. (164) Yamashita,Y.,Ashizawa,T.,Morimoto,M., Hosomi,J.,andNakano, H. (1992) Antitumor quinolones with mammalian topoisomerase I1 mediated DNA cleavage activity. Cancer Res. 52, 2818-2822. (165) Shen, L. L., Kohlbrenner, W. E., Weigl, D., and Baranowski, J. (1989) Mechanism of quinolone inhibition of DNA gyrase: A cooperative drug-DNA binding model. J.Biol. Chem. 264,29732978. (166) Willmott, C. J., and Maxwell, A. (1993) A single point mutation in DNA gyrase A protein greatly reduces binding of fluoroquinolones to the gyraseDNA complex. Antimicrob. Agents Chemother. 37, 126-127. (167) Hertzberg, R. P., Caranfa, M. J., and Hecht, S. M. (1989) On the mechanism of topoisomerase I inhibition by camptothecin: Evidence for binding to an enzymeDNA complex. Biochemistry 28, 4629-4638. (168) Traganos, F., Ardelt, B., Halko, N., Bruno, S., and Darzynkiewicz, Z. (1992) Effectaof genistein on the growth and cell cycle progression of normal human lymphocytes and human leukemic MOLT-4 and HL-60 cells. Cancer Res. 52, 6200-6208. (169) Zimmer, C., Storl, K., and Storl, J. (1990) Microbial DNA topoisomerases and their inhibition by antibiotics. J. Basic Microbiol. 30, 209-224. (170) Hooper, D. C., and Wolfson, J. S. (1991) Fluoroquinolone antimicrobial agents. New Engl. J. Med. 324, 384-394. (171) Maxwell, A. (1992) The molecular basis of quinolone action. J. Antimicrob. Chemother. 30, 409-414. (172) Barrett, J. F., Gootz, T. D., McGuirk, P. R., Farrell, C. A., and Sokolowski, S. A. (1989) Use of in vitro topoisomerase I1 assays for studying quinolone antibacterial agents. Antimicrob. Agents Chemother. 33, 1697-1703. (173) Gootz, T. D., Barrett, J. F., and Sutcliffe, J. A. (1990) Inhibitory effects of quinolone antibacterial agents on eucaryotic topoisomerases and related test systems. Antimicrob. Agents Chemther. 34, 8-12. (174) Wentland, M. P., Lesher, G. Y., Reuman, M., Pilling, G. M., Saindane, M. T., Perni, R. B., Eissenstat, M. A., Weaver, J. D., Rake, J. B., and Coughlin, S. A. (1991) Mammalian topoisomerase I1 inhibitory activity of l&bridged-7-(2,6-dimethy1-4-pyridinyl)3-quinolinecarboxylic acids. Roc. Am. Assoc. Cancer. Res. 32,336. (175) Kohlbrenner, W. E., Wideburg, N., Weigl, D., Saldivar, A., and Chu, D. T. W. (1992) Induction of calf thymus topoisomerase IImediated DNA breakage by the antibacterial isothimloquinolones, A-65281and A-65282. Antimicrob. Agents Chemother. 36,81-86. (176) Robinson, M. J., Martin, B. A., Gootz, T. D., McGuirk, P. R., and Osheroff, N. (1992) Effects of novel fluoroquinoloneson the catalytic activities of eukaryotic topoisomerase 11: Influence of the C-8 fluorine group. Antimicrob. Agents Chemother. 36, 751-756. (177) Wentland, M. P., Lesher, G. Y., Reuman, M., Gruett, M. D., Singh, B., Aldous, S. C., Dorff, P. H., Rake, J. B., and Coughlin, S. A. (1992) Relationships of mammalian topoisomerase I1 inhibitory potency to the structure of 6,&difluoro-1,4-dihydro-4-oxo-3-quinolonecarboxylic acid. h o c . Am. Assoc. Cancer. Res. 33, 431. (178) Elsea, S. H., McGuirk, P. R., Gootz, T. D., Moynihan, M., and Osheroff, N. (1993) Drug features that contribute to the activity of quinolones against mammalian topoisomerase I1 and cultured cells: Correlation between the enhancement of enzyme-mediated DNAcleavage invitroandcytotoxicpotential. Antimicrob. Agents Chemother., in press. (179) Froelich-Ammon, S. J., McGuirk, P. R., Gootz, T. D., Jefson, M. R., and Osheroff, N. (1993) Novel 1-&bridged chiral quinolones with activity against topoisomerase I 1 Stereospecificity of the eukaryotic enzyme. Antimicrob. Agents Chemother. 37,646-651. (180) Jolivet, J.,Cowan, K. H.,Curt,G. A.,Clendeninn,N. J.,and Chabner, B. A. (1983) The pharmacology and clinical use of methotrexate. New Engl. J. Med. 309, 1094-1104. (181) Werkheiser, W. C. (1963) The biochemical, cellular, and uharmacological action and effects of the folic acid antagonists.- Cancer Res. 23, 1277-1285. (182) Donehower, R. C., Myers, C. E., and Chabner. B. A. (1979) New developments on the mechanism of action of antineoplastic drugs. Life Sci. 25, 1-14. (183) Alt, F. W., Kellems. R. E., Bertino, J. R., and Schimke, R. T. (1978) Selective multiplication of dihydrofolate in methotrexate-resistant variants of cultured murine cells. J. Biol. Chem. 253,1357-1370.
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 595 (184) Pommier, Y., Minford, J. K., Schwartz, R. E., Zwelling, L. A,, and Kohn, K. W. (1985) Effects of the DNA intercalators 4’49acridiny1amino)methanesulfon-m-anisidideand 2-methyl-9-hydroxyellipticinium on topoisomerase I1 mediated DNA strand cleavage and strand passage. Biochemistry 24, 6410-6416. (185) Gale, K. C., and Osheroff, N. (1990) Uncoupling the DNA cleavage and religation activities of topoisomerase I1 with a single stranded nucleic acid substrate: Evidence for an active enzyme-cleaved intermediate. Biochemistry 29,9538-9545. (186) Andersen, A. H., Sorensen, B. S., Christiansen, K., Svejstrup, J. Q., Lund, K., and Westergaard, 0. (1991)Studies of the topoisomerase 11-mediated cleavage and religation reactions by use of a suicidal double-stranded DNA substrate. J. Biol. Chem. 266,9203-9210. (187) Zwelling, L. A., Michaels, S., Erickson, L. C., Ungerleider, R. S., Nichols, M., and Kohn, K. W. (1981) Protein-associated deoxyribonucleic acid strand breaks in L1210 cells treated with the deoxyribonucleic acid intercalating agents 4’-(9-acridinylamino)methanesulfon-m-anisidideand adriamycin. Biochemistry 20, 6553-6563. (188) Pommier, Y., Schwartz, R. E., Kohn, K. W., and Zwelling, L. A. (1984) Formation and rejoining of deoxyribonucleic acid doublestrand breaks induced in isolated cell nuclei by antineoplastic intercalating agents. Biochemistry 23, 3194-3201. (189) Pommier, Y., Schwartz, R. E., Zwelling, L. A., and Kohn, K. W. (1985) Effects of DNA intercalating agents on topoisomerase I1 induced DNA strand cleavage in isolated mammalian cell nuclei. Biochemistry 24, 6406-6410. (190) Yang, L., Rowe, T. C., and Liu, L. F. (1985) Identification of DNA topoisomerase I1 as an intracellular target of antitumor epipodophyllotoxins in simian virus 40-infected monkey cells. Cancer Res. 45,5872-5876. (191) Yang, L., Rowe, T. C., Nelson, E. M., and Liu, L. F. (1985) In vivo mapping of DNA topoisomerase 11-specificcleavage sites in SV40 chromatin. Cell 41, 127-132. (192) Minford, J., Pommier, Y., Filipski, J., Kohn, K. W., Kerrigan, D., Mattern, M., Michaels, S., Schwartz, R., and Zwelling, L. A. (1986) Isolation of intercalator-dependent protein-linked DNA strand cleavageactivity from cell nuclei and identification as topoisomerase 11. Biochemistry 25, 9-16. (193) Schneider, E., Lawson, P. A., and Ralph, R. K. (1989) Inhibition of protein synthesis reduces the cytotoxicity of 4’-(9-acridinylamino)methanesulfon-m-anisididewithout affecting DNA breakage and DNA topoisomerase I1 in a murine mastacytoma cell line. Biochem. Pharmacol. 38, 263-269. (194) DArpa, P., Beardmore, C., and Liu, L. F. (1990) Involvement of nucleic acid synthesis in cell killing mechanisms of topoisomerase I1 poisons. Cancer Res. 50, 6919-6924. (195) Zhang, H., D’Arpa, P., and Liu, L. F. (1990) A model for tumor cell killing by topoisomerase I1 poisons. Cancer Cells 2, 23-27. (196) Nitiss, J. L., and Wang, J. C. (1988) DNA topoisomerase targeting antitumor drugs can be studied in yeast. Roc. Natl. Acad. Sci. U.S.A. 85, 7501-7505. (197) Bae, Y.-S., Chiba, M., Ohira, M., and Ikeda, H. (1991) A shuttle vector for analysis of illegitimate recombination in mammalian cells: Effects of DNA topoisomerase inhibitors on deletion frequency. Gene 101, 285-289. (198) Elsea, S. H., Osheroff, N., and Nitiss, J. L. (1992) Cytotoxicity of quinolones toward eukaryotic cells: Identification of topoisomerase I1 as the primary cellular target for the quinolone CP-115,953 in yeast. J. Biol. Chem. 267, 13150-13153. (199) Han, Y.-H., Austin, M. J. F., Pommier, Y., and Povirk, L. F. (1993) Small deletion and insertion mutations induced by the topoisomerme I1inhibitor teniposide in CHO cells and comparison with sites of drug-stimulated DNA cleavage in vitro. J.Mol. Biol.229, 52-66. (200) Renault, G., Malvy, C., Venegas, W., and Larsen, A. K. (1987) In vivo exposure to four ellipticine derivatives with topoisomerase inhibitory activity results in chromosome clumping and sister chromatid exchange in murine bone marrow cells. Toxicol. App. Pharmacol. 89, 281-286. (201) Andersson, H. C., and Kihlman, B. A. (1989) The production of chromosomal alterations in human lymphocytes by drugs known to interfere with the activity of DNA topoisomerase 11. I. m-AMSA. Carcinogenesis 10, 123-130. (202) DeVore, R., Whitlock, J., Hainsworth, T., and Johnson, D. (1989) Therapy-related acute nonlymphocytic leukemia with monocytic featuresand rearrangement of chromosome llq. Ann. Intern. Med. 110, 740-742. (203) LBnn, U., Lonn, S., Nylen, U., and Winbald, G. (1989) Altered formation of DNA in human cells treated with inhibitors of DNA topoisomerase I1 (etoposide and teniposide). CancerRes. 49,62026207.
596 Chem. Res. Toxicol., Vol. 6, No. 5, 1993 (204) Charron, M., and Hancock, R. (1991) Chromosome recombination and defective genome segregation induced in Chinese hamster cells by the topoisomerase I1 inhibitor VM-26. Chromosoma 100,97102. (205) Kaufman, S. (1989) Induction of endonucleolytic DNA cleavagein human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: A cautionary note. Cancer Res. 49,5870-5878. (206) Hickman, J. A. (1992) Apoptosis induced by anticancer drugs. Cancer Metastasis Rev. 11,121-139. (207) Roy, C., Brown, D. L., Little, J. E., Valentine, B. K., Walker, P. R., Sikoreka, M., LeBlanc, J., and Chaly, N. (1992) The topoisomerase I1inhibitor teniposide (VM-26) induces apoptosis in unstimulated mature murine lymphocytes. Erp. Cell Res. 200,416-424. (208) Onishi, Y., Azuma, Y., Sato, Y., Mizuno, Y., Tadakuma, T., and Kizaki, H. (1993) Topoisomerase inhibitors induce apoptosis in thymocytes. Biochim. Biophys. Acta 1175, 147-154. (209) Kreuzer, K. N., and Cozzarelli,N. R. (1979)Escherichia coli mutants thermosemitive for deoxyribonucleicacid gyrase subunit A: Effects on deoxyribonucleic acid replication, transcription, and bacteriophage growth. J.Bacteriol. 140, 424-435. (210) Ross, W. E., Glaubiger, D. L., and Kohn, K. W. (1978) Proteinassociated DNA breaks in cells treated with adriamycin or ellipticine. Biochim. Biophys. Acta 519, 23-30. (211) Roes, W. E., Glaubiger, D. L., and Kohn, K. W. (1979) Qualitative and quantitative aspects of intercalator-induced DNA strand breaks. Biochim. Biophys. Acta 562, 41-50. (212) Long, B. H. (1987) Structure-activity relationships of podophyllin congenersthat inhibit topoisomerase11. Natl. Cancer h t .Monogr. 4, 123-127. (213) Gupta, R. S. (1983) Genetic, biochemical, and cross-resistance studies with mutants of Chinese hamster ovary cells resistant to the anticancer drugs, VM-26and VP16-213. Cancer Res. 43,15681574. (214) Beck, W. T., Cirtain, M. C., Danks, M. K., Felsted, R. L., Safa, A. R., Wolverton, J. S., Suttle, D. P., and Trent, J. M. (1987) Pharmacological,molecular, and cytogenetic analysis of “atypical“ multidrug-resistant human leukemic cells. Cancer Res. 47,54555460. (215) Beran, M., and Andersson, B. S. (1987) Development and characterization of a human myelogenous leukemia cell line resistant to 4’-(9-acridinylamino)methanesulfon-m-anisidide. Cancer Res. 47,1897-1904. (216) Danks, M. K., Yalowich, J. C., and Beck, W. T. (1987) Atypical multiple drug resistance in a human leukemic cell line selected for resistance to teniposide (VM-26). Cancer Res. 47, 1297-1301. (217) Danks, M. K., Schmidt, C. A., Cirtain, M. C., Suttle, D. P., and Beck, W. T. (1988) Altered catalytic activity of and DNA cleavage by DNA topoisomerase I1 from human leukemic cells selected for resistance to VM-26.Biochemistry 27,8861-8869. (218) Sullivan, D. M., Latham, M. D., Rowe, T. C., andRoss, W. E. (1989) Purification and characterization of an altered topoisomerase I1 fromadrug-resistant Chinesehamster ovary cell line. Biochemistry 28,5680-5687. (219) Zwelling, L. A,, Hinds, M., Chan, D., Mayes, J., Sie, K. L., Parker, E., Silberman, L., Radcliffe, A., Beran, M., and Blick, M. (1989) Characterization of an amsacrine-resistant line of human leukemia cells. J.Biol. Chem. 264, 16411-16420. (220) Zwelling, L. A., Mayes, J., Hinds, M., Chan, D., Altschuler, E., Carroll, B., Parker, E., Deisseroth, K., Radcliffe, A., Seligman, M., Li, L., and Farquhar, D. (1991) Cross-resistant human leukemia line to topoisomerase I1 reactive DNA intercalating agents. Evidence for two topoisomerase I1 directed drug actions. Biochemistry 30,4048-4055. (221) Jannatipour, M., Liu, Y. X., and Nitiss, J. L. (1993) The top2-5 mutant of yeast topoisomerase I1 encodes an enzyme resistant to etoposide and mAMSA. J. Biol. Chem., in press. (222) Nitiss, J. L., Hsiung, Y., Jannatipour, M., and Wu, H. (1993) Characterization of top2 mutations conferring resistance to topoisomerase I1 targeting agents. Roc. Am. Assoc. Cancer Res. 34, 426. (223) Patel, S.,Austin, C. A., and Fisher, L. M. (1990) Development and properties of an etoposide-resistant human leukemic CCRF-CEM cell line. Anticancer Drug Des. 5, 149-157. (224) Bugg, B. Y., Danks, M. K., Beck, W. T., and Suttle, D. P. (1991) Expression of a mutant DNA topoisomerase I1 in CCRF-CEM human leukemic cells selected for resistance to teniposide. Roc. Natl. Acad. Sci. U.S.A. 88,7654-7658. (225) Danks, M. K., Warmouth, M. R., Friche, E., Granzen, B., Bugg, B. Y., Harker, W. G., Zwelling, L. A., Futscher, B. W., Suttle, D. P., and Beck, W. T. (1993)Single-strand conformationalpolymorphism analysis of the M, 170,000 isozyme of DNA topoisomerase I1 in human tumour cells. Cancer Res. 53,1373-1379. (226) Lee, MA., Wang, J. C., and Beran, M. (1992) Two independent amsacrine-resistant leukemia cell lines share an identical point
Corbett and Osheroff mutation in the 170 kDa form of human topoisomerase 11. J.Mol. Biol. 223, 837-843. (227) Chan, V. T.-W., Ng, S., Eder, J. P., and Schnipper, L. E. (1993) Molecular cloning and identification of a point mutation in the topoisomerase I1 cDNA from an etoposide-resistant Chinese hamster ovary cell line. J. Biol. Chem. 268,2160-2165. (228) Patel, S., and Fisher, L. M. (1993) Novel skeleton and genetic characterization of an etoposide-resistant human leukemic CCRFCEM cell line. Br. J. Cancer 67, 456-463. (229) Nitiss, J. L., Liu, X.-Y., Harbury, P., Jannatipour, M., Wasserman, R., and Wang,J. C. (1992)Amsacrine and etopoeidehypersensitivity of yeast cells overexpressingDNA topoisomerase 11. Cancer Res. 52,4467-4472. (230) Nitiss, J. L., Liu, X.-Y., and Hsiung, Y. (1993) A temperature sensitive topoisomerase I1 allele confers temperature dependent drug resistance on amsacrine and etoposide: A genetic system for determining the targets of topoisomerase I1 inhibitors. Cancer Res. 53, 89-93. (231) Davies, S. M., Robson, C. N., Davies, S. L., and Hickson, I. D. (1988)Nuclear topoisomerase I1 levels correlate with the sensitivity of mammalian cella to intercalating agents and epipodophyllotoxins. J.Biol. Chem. 263, 17724-17729. (232) Potmesil, M., Hsiang, Y.-H., Liu, L. F., Bank, B., Grossberg, H., Kirschenbaum, S., Forlenzar, T. J., Penziner, A., Kanganis, D., (1988)Resistanceofhuman Knowles, D.,Traganos,F.,andSilber,R. leukemic and normal lymphocytes to drug-induced DNA cleavage and low levels of DNA topoisomerase 11. Cancer Rea. 48, 35373543. (233) Deffie, A. M., Batra, J. K., and Goldenberg, G. J. (1989) Direct correlation between DNA topoisomeraseI1activity and cytotoxicity in Adriamycin-sensitive and -resistant P388 leukemia cell lines. Cancer Res. 49, 58-62. (234) Friche, E., Danks, M. K., Schmidt, C. A., and Beck, W. T. (1991) Decreased DNA topoisomeraseI1in daunorubicin-resistant Ehrlich ascites tumor cells. Cancer Res. 51,4213-4218. (235) Fry, A. M., Chresta, C. M., Davies, S. M., Walker, M. C., Harris, A. L., Hartley, J. A,, Masters, J. R. W., and Hickson, I. D. (1991) Relationship between topoisomerase I1level and chemosensitivity in human tumor cell lines. Cancer Res. 51,65924595. (236) Webb, C. D., Latham, M. D., Lock, R. B., and Sullivan, D. M. (1991)Attenuated topoisomerase I1content directly correlates with a low level of drug resistance in a Chinese hamster ovary cell line. Cancer Res. 51,6543-6549. (237) Takano, H., Kohno, K., Ono, M., Uchida, Y., and Kuwano, M. (1991) Increased phosphorylation of DNA topoisomerase I1 in etoposide-resistant mutants of human cancer KB cells. Cancer Res. 51, 3951-3957. (238) Ganapathi, R., Zwelling, L., Constantinou, A., Ford, J., and Grabowski, D. (1993) Altered phosphorylation, biosynthesis, and degradation of the 170 kDa isoformof topoisomeraseI1in amsacrineresistant human leukemia cells. Biochem. Biophys. Res. Commun. 192, 1274-1280. (239) Capranico,G.,Kohn,K. W.,andPommier,Y. (1990)Localsequence requirements for DNA cleavage by mammalian topoisomerase I1 in the presence of doxorubicin. Nucleic Acids Res. 18,66114619. (240) Pommier, Y., Capranico, G., Orr, A., and Kohn, K. (1991) Local base sequence preferences for DNA cleavage by mammalian topoisomerase I1in the presence of amsacrineorteniposide. Nucleic Acids Res. 19, 5973-5980. (241) Capranico, G., Isabella, P. D., Tinelli, S., Bigioni, M., and Zunino, F. (1993) Similar sequence specificity of mitoxantrone and VM-26 stimulation of in vitro DNA cleavage by mammalian DNA topoisomerase 11. Biochemistry 32, 3038-3046. (242) Gerwitz, D. A. (1991) Does bulk damage to DNA explain the cytostatic and cytotoxic effects of topoisomerase I1 inhibitors? Biochem. Pharmacol. 42, 2253-2258. (243) Hsiang, Y.-H., and Liu, L. F. (1989) Evidence for the revereibility of cellular DNA lesions induced by mammalian topoisomerase I1 poisons. J. Biol. Chem. 264, 9713-9715. (244) Zechiedrich,E. L., Christiansen, K., Andersen, A. H., Westergaard, O., and Osheroff, N. (1989) Double-stranded DNA cleavage religation reaction of eukaryotic topoisomerase I 1 Evidence for a nicked DNA intermediate. Biochemistry 28, 6229-6236. (245) Andersen, A. H., Smensen, B. S., Christiansen, K., Svejstrup, J. Q., Lund, K., and Westergaard, 0.(1991) Studieaof the topoisomerase 11-mediatedcleavage and religation reactions by use of a suicidal double-stranded DNA substrate. J. Biol. Chem. 266,9203-9210. (246) Smensen, B. S., Sinding, J., Andersen, A. H., Alsner, J., Jensen, P. B., and Westergaard, 0. (1992) Mode of action of topoisomerase 11-targeting agents at a specific DNA sequence: Uncoupling the DNA binding, cleavage and religation events. J.Mol. Biol. 228, 778-786.
Invited Reviews
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 597
(247) Osheroff, N. (1989) Effect of antineoplastic agents on the DNA
(252) Yoshida, Y., Bogaki, M., Nakamura, M., and Nakamura, S. (1990)
cleavagelreligation equilibrium of eukaryotic topoisomerase I1 Inhibition of DNA religation by etoposide. Biochemistry 28,6157-
Quinolone resistance-determining region in the DNA gyrase gyrA geneof Escherichia coli. Antimicrob. Agents Chemother. 34,1271-
6160. (248) Robinson, M. J., and Osheroff, N. (1990) Stabilization of the
1272. (253) Hallett, P., and Maxwell, A. (1991) Novel quinolone resistance
topoisomerase 11-DNA cleavage complex by antineoplastic drugs: Inhibition of enzyme-mediated DNA religation by 4’-(9-acridinylamino)methanesulfon-m-anisidide.Biochemistry 29,2511-2515. (249) Corbett, A. H., Hong, D., and Osheroff, N. (1993) Exploiting mechanistic differences between drug classes to define functional drug interaction domainson topoisomeraseI1 Evidencethat several diverse DNA cleavage-enhancing agents share a common site of action on the enzyme. J. Biol. Chem. 268,14394-14398. (250) Robinson, M. J., Corbett, A. H., Osheroff, N. (1993) Effects of topoisomerase 11-targeteddrugs on enzyme-mediatedDNAcleavage and ATP hydrolysis: Evidencefor distinct drug interaction domains on topoisomerase 11. Biochemistry 32, 3638-3643. (251) Fisher, L. M., Lawrence, J. M., Josty, J. M., Hopewell, R., Maregerrison, E. E. C., and Cullen, M. E. (1989) Ciprofloxacin and the fluoroquinolones: New concepts on the mechanism of action and resistance. Am. J. Med. 87 (Suppl. 5A), 25-85.
mutationsoftheEscherichia coli DNAgyraseA protein: Enzymatic analysis of the point mutations. Antimicrob. Agents Chemother. 35,335-340. (254) Yoshida, H., Bogaki, M., Nakamura, M., Yamanaka, L. M., and Nakamura, S. (1991) Quinolone resistance-determining region in
the DNAgyrasegyrB gene ofEscherichia coli. Antimicrob. Agents Chemother. 35, 1647-1650. (255) Stevenson, R. L. (1886) The Strange Case of Dr. Jekyll and Mr. Hyde, Longmans, Green, & Co., London. (256) Jeggo, P. A., Caldecott, K., Pidsley, S., and Banks, G. R. (1989) Sensitivity of Chinese hamster ovary mutants defective in DNA double strand break repair to topoisomerase I1 inhibitors. Cancer Res. 49, 7057-7063. (257) Caldecott, K., Banks, G., and Jeggo, P. (1990) DNA double-strand break repair pathways and cellular tolerance to inhibitors of topoisomerase 11. Cancer Res. 50, 5778-5783.