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Culprit and victim – DNA topoisomerase II
Review
DNA topoisomerase II in oncology
Culprit and victim – DNA topoisomerase II Udo Kellner, Maxwell Sehested, Peter B Jensen, Frank Gieseler, and Pierre Rudolph
The phylogenetic antiquity of DNA topoisomerases indicates their vital function. Structure and maintenance of genomic DNA depend on the activity of these enzymes, and without them DNA replication and cell division are impossible. Topoisomerase II␣ has therefore become the main target of many antitumour therapy regimens, even though the exact mechanism of cell killing remains elusive. The success of this approach is limited by the development of spontaneous resistance, and druginduced DNA damage can increase malignancy. Nevertheless, the combined use of topoisomeraseinhibiting drugs with different mechanisms of action promises to improve particular treatment designs. The degree of topoisomerase II expression in tumours may predict the clinical course and responsiveness to therapy. Lancet Oncol 2002; 3: 235–43
Topoisomerases are enzymes that can modify (isomerise) the tertiary structure of DNA without changing its primary structure,1 which is determined by the nucleotide sequence. The high degree of conservation of these enzymes among prokaryotes and eukaryotes2 indicates an essential role in cell biology. In human beings, two classes of topoisomerases are well characterised—type I and type II, of which the latter will be discussed in this review. All physiological functions of DNA depend on its tertiary configuration, and therefore the activity of topoisomerase is of vital importance for all DNA-containing cells (figure 1). Because its structure is a double helix, DNA is under tortional stress that results in multiplex twisting of the molecule. To be processed for replication or gene expression, the supercoiled DNA must become accessible to polymerases or components of the transcription machinery. This change requires relaxation and untangling of the intertwined DNA strands, which are the typical tasks of DNA topoisomerases. The anthracyclines and epipodophyllotoxins have been mainstays in medical oncology since the 1960s, and were eventually shown to target DNA topoisomerase II. Poisons of the enzyme and related compounds were among the earliest drugs used in cancer therapy; the first written mention of podophyllotoxins in cancer treatment dates from the 8th century.3 Efforts made during the 1950s to increase the efficacy of the native substances by minor chemical modifications led to the development of the first two semisynthetic (epi-)podophyllotoxin derivatives, etoposide and teniposide, which unlike the parent podophyllotoxins do not inhibit microtubule assembly but do inhibit topoisomerase II. Clinical trials started in 1971 and showed activity in acute myeloid leukaemia (AML), Hodgkin’s disease, non-Hodgkin lymphoma, and
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ATPase
T
ATP
B 2
A
G
1 3
ATP+Pi
(CC1)
5 4 (CC2)
3'
Figure 1. Proposed structure and catalytic cycle of topoisomerase II. One DNA strand ‘G’ (gate) is cleaved, allowing the passage of another intact DNA strand ‘T’ (transport).The enzyme is a homodimer consisting of three functional segments, an ATPase (top), a cleavage (B), and a C-terminal (A) part. The topoisomerase-II poisons act by stabilising stage 3 or 4, where the DNA strand is cleaved. The catalytic inhibitors act either at stage 1 (chloroquine and aclarubicin) or at stage 5 (bisdioxopiperazines such as dexrazoxane). Figure modified from reference 1.
carcinomas of the lung, stomach, breast, and ovaries. Etoposide is now a standard agent in treatment of small-cell lung cancer and testicular cancer (table 1). The antibiotic group of anthracyclines, which are fermentation products of Streptomyces peuceus, entered clinical oncology in the 1960s. However, these antibiotics were not recognised to be directed against topoisomerase II until 1984.4 Anthracyclines are now widely used, the most commonly is doxorubicin which can be used for for lymphomas, breast cancer, sarcomas, Kaposi’s sarcoma, and leukaemias. Epirubicin, which may be less cardiotoxic than doxorubicin, is extensively used in Europe for treatment of breast cancer, and it has recently been approved by the US Food and Drug Administration for adjuvant therapy of breast cancer (table 1). Daunorubicin and idarubicin are used in acute leukaemias. Other clinically applied topoisomerase II-poisons are mitoxantrone, an anthracenedione for acute leukaemias, breast and ovarian cancers, bladder cancer, and lymphomas, and amsacrine in acute leukaemia. UK is at the Department of Pathology, University of Magdeburg, Germany. MS and PBJ are at the Finsen and Laboratory Centres, Rigshospitalet, Copenhagen, Denmark. FG is at the Section of Hematology/Oncology, Department of Internal Medicine, University Hospital of Kiel, Germany. PR is at the Department of Pathology, University of Kiel, Germany. Correspondence: Dr Udo Kellner, Institut für Pathologie, Otto-von-Guericke Universität, 39120 Magdeburg, Germany. Tel: +49 391 67 14 295. Fax: +49 391 67 190 646. Email: [email protected]
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Review
DNA topoisomerase II in oncology
Table 1. Overview of the topoisomerase-II-targeting drugs in current clinical use and the range of successful applications Drug
Class
Applications
Topoisomerase-II␣ poisons Epirubicin
Anthracycline
Adjuvant breast cancer
Doxorubicin
Anthracycline
Idarubicin
Anthracycline
Non-Hodgkin lymphoma, chronic lymphocytic leukaemia, Hodgkin’s disease, multiple myeloma, acute leukaemia, small-cell lung cancer, breast, ovarian, and bladder cancers, sarcomas Acute leukaemia
Daunorubicin
Anthracycline
Acute leukaemia
Mitoxantrone
Antracendione
Acute leukaemia, breast, ovarian, and bladder cancers
Amsacrine
Ansidide
Acute leukaemia
Etoposide
Epipodophyllotoxin
Small-cell lung cancer, testicular cancer, ovarian tumours, acute myeloid leukaemia, non-Hodgkin lymphoma
Teniposide
Epipodophyllotoxin
Small-cell lung cancer
Topoisomerase-II␣ catalytic inhibitors Aclarubicin
Anthracycline
Acute myeloid leukaemia
Sobuzoxane
Bisdioxopiperazine
T-cell lymphomas and leukaemia
Dexrazoxane
Bisdioxopiperazine
Central nervous system metastases, anthracycline extravasation
Several resistance mechanisms limit the clinical success of topoisomerase-targeting drugs. Moreover, secondary malignant disorders can arise after topoisomerase-directed treatment regimens. To understand the benefits and disadvantages of these therapeutic approaches, we will take a closer look at the mechanisms of action of topoisomerase II, its role in neoplasia, and the biology of pharmacological topoisomerase inhibition.
Mechanism of action, regulation, and physiological functions Topoisomerase II is the only enzyme able to cleave and religate double-stranded DNA. This enzyme acts in the relaxation of DNA supercoils that accumulate during gene transcription and along with the progression of the replication fork. Moreover, only topoisomerase II can carry out the decatenation of replicated circular double-stranded DNA, and it is obligatorily involved in the remodelling of chromatin during mitosis.5 There are two highly homologous isoforms of topoisomerase II in human beings, which are encoded by different genes. The gene for topoisomerase II␣ is on chromosome 17q21–22 and encodes a 170 kDa protein.6 The topoisomerase II gene, encoding a 180 kDa protein, is on chromosome 3q24.7 The function of topoisomerase II requires dimerisation, mainly in form of homodimers, but heterodimers occur.8 Both enzymes are ATP-dependent endonucleases and ligases that cut both DNA strands and covalently bind to the new DNA 5⬘ end. The catalytic action starts with an energyindependent endonucleic DNA cut and a 5⬘ covalent linking (figure 1). The double strand break is fixed by disulphide bonds bridging the topoisomerase II dimer,1 which constitutes the so-called cleavable complex. Under ATP hydrolysis, the nicked DNA double strand is (re)ligated, and the topoisomerase II dimer eventually dissociates from the DNA. This mode of action explains why topoisomerase II activity is needed at several points in DNA metabolism, such as transcription, replication, and chromosome condensation and segregation.9 In addition, a role for topoisomerase II in nucleotide excision repair has been discussed.10,11
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Several complex mechanisms modulate the activity of topoisomerase II, including transcriptional and posttranscriptional regulation, and post-translational modifications such as phosphorylation, protein interactions, and proteasome-mediated degradation.12 The expression of topoisomerase II␣ is regulated with the cell cycle, whereas that of the II form is not.13 In proliferating cells, topoisomerase II␣ becomes detectable in late G1 phase, and the amount gradually increases to peak in G2/M (figures 2 and 3). The induction of quiescence or terminal differentiation, by contrast, results in striking downregulation of expression of this enzyme. The main transcription factor that brings about transactivation of topoisomerase II␣, nuclear factor Y, binds to the proximal inverted CCAAT boxes in the topoisomerase II␣ promoter region with constant avidity throughout the cell cycle.14 This apparent paradox is explained by the observation that the association of nuclear factor Y with histone acetyltransferase greatly increases topoisomerase II␣ promoter activity, downregulation during G0/G1 being achieved by trichostatin-mediated histone deacetylation.14 The dependency on the cell-cycle phase of topoisomerase II␣ expression supports the concept that the enzyme has a major role in cell proliferation, but there is no compelling evidence indicating involvement of the  isoform. The physiological functions of the latter form are poorly understood, although the two enzymes share the same mechanism of action and target similar cleavage sites.15 Transgenic cell lines lacking topoisomerase II apparently remain viable.16 By contrast, TOP2 knock-out mice die shortly after birth from respiratory insufficiency despite initially normal development during the embryonic period; motor axons fail to contact skeletal muscles.17 Although this finding does not elucidate the physiological role of topoisomerase II, it points to a vital function that is apparently unrelated to cell proliferation. The  isoenzyme may rescue the function of topoisomerase II␣ in proliferating cells when the latter is expressed at inappropriately low concentrations, mutated, or inhibited pharmacologically. This idea is supported by the ability of
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DNA topoisomerase II in oncology
topoisomerase II to form heterodimers, and by evidence suggesting an evolution of the two isoforms through gene duplication.18 Beyond gene expression, phosphorylation of topoisomerase II␣ is required to increase its activity. The main factor involved in this process is casein kinase II, although recent evidence indicates involvement of the mitogen-activated protein kinase extracellular signal-regulated kinase pathway,19 in accordance with an induction of topoisomerase activity in response to mitogenic signals.20 Despite a direct interaction, the mitotic CDC2 kinase (CDK1), which forms together with cyclin B the mitosis-promoting factor MPF, apparently does not phosphorylate Figure 2. Immunohistochemical staining of a small-cell carcinoma of the lung with KiS5. In this topoisomerase II␣ while stimulating tumour type, which can be successfully treated with etoposide and teniposide, topoisomerase II␣ is its catalytic activity and its binding to expressed in up to 70% of the cells. Only cycling cells (G through mitosis) are labelled, with variable intensity reflecting differing expression. The staining pattern is similar to that obtained with the chromatin.21 This newly recognised proliferation marker Ki67. mechanism points to cooperation of the two proteins in chromatin remodelling and explains the breaks.30 This effect, which seemingly is not brought about requirement for topoisomerase II␣ in chromosome by other 14-3-3 proteins, seems to be attributable to a decrease in the DNA-binding activity of topoisomerase II␣, condensation. Activity and expression of topoisomerase II are further suggesting an additional mechanism of physiological influenced by interaction with two master regulators of the regulation. cell cycle, p53, and the retinoblastoma susceptibility gene product, pRB. The stress-activated tumour suppressor Topoisomerase II and tumour biology protein p53 can downregulate topoisomerase II␣ expression In view of the many roles of topoisomerase II in DNA by transcriptional regulation22 and modulate the activity of processing, an oncogenic effect associated with the activity of both the ␣ and  isoenzymes by direct protein interaction.23 these enzymes might be expected. Defective function of This mechanism is probably designed to halt replication in topoisomerase II engendering inappropriate DNA breaks the case of DNA damage. p53 nevertheless seems to exert a can lead to incorrect strand recombination, and dual function, because DNA damage provoked by various chromosomal translocations can result from incorrect agents such as ionising radiation (with the exception of segregation during mitosis. Rearrangements of the genome ultraviolet photoproducts) greatly stimulates the cleavage commonly occur by way of illegitimate recombination activity of topoisomerase II,24 which is mediated by the p53- between DNA loop anchorage sites, and topoisomerase II dependent stress-induced Gadd45 protein.25 Induction of has been implicated in this process.31 Although DNA doublewild-type p53 also seems to result in a large increase in strand breaks are likely to induce a lethal phenotype topoisomerase II content in some cell types.26 The resulting in a mitotic catastrophe in most instances, some biological significance of this process is not clear. In addition cells may survive and eventually acquire genomic to p53, pRB physically interacts with topoisomerase II␣ and aberrations such as chromosomal translocations or DNA efficiently inhibits its activity,27 which discloses another losses that promote malignancy.32,33 Topoisomerase II might conceivably function as a feature of the pRB-mediated negative cell cycle regulation at recombinase, as topoisomerase I does, since substitution of a the G1/S boundary.28 Another interesting facet of topoisomerase II␣ has lately single aminoacid in the restriction endonuclease, NaeI, is emerged—its interaction with a protein from the 14-3-3 enough to elicit both topoisomerase and recombinase family. Although their exact function is largely unknown, activity.34 More probably, however, strand breaks generated mounting evidence suggests that these proteins are involved by unscheduled action of topoisomerases may trigger in several important biological processes such as signal recombination pathways as the cells attempt to repair the transduction, cell-cycle control, apoptosis, and stress damage, or recombinations may arise from spontaneous endresponse in that they act as adaptor molecules stimulating to-end fusion of unprotected DNA strands.35 As a corollary, protein-protein interactions, regulate the subcellular the essentially cytotoxic effects of topoisomerase poisons can location of proteins, and activate or inhibit enzymes.29 We be mutagenic when cells survive the genomic damage.36 Increased expression of topoisomerase II, a common observed that 14-3-3⑀ negatively affects the capacity of etoposide to produce topoisomerase-mediated DNA strand occurrence in neoplasia (figures 2 and 3), is by no means 1
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DNA topoisomerase II in oncology
the other, which would explain the concurrent overexpression of the two gene products in breast cancer.37 Topoisomerase II␣ is a reliable proliferation marker, because the cellular content of the enzyme directly mirrors the proliferative activity of the cells. In 1992, Sampson and coworkers described a monoclonal antibody with affinity for a nuclear antigen in proliferating cells, Ki-S1, which seemed to provide useful prognostic information in breast carcinoma.40 This marker was also found to be a good indicator of malignancy in melanocytic lesions.41 Eventually, the antigen recognised by this antibody was shown to be Other topoisomerase II␣.20 monoclonal antibodies to this enzyme have since been developed, which yield clearer and more reproducible results than the original marker (figure 2).20 Figure 3. Immunohistochemical staining of Hodgkin’s cells and lymphocytes (nodular sclerosis). In With these antibodies, we showed in this cancer type, which can be successfully treated with regimens such as ABVD (doxorubicin, extensive retrospective studies, bleomycin, vinblastine, dacarbacine), CEP (cyclophosphamide, etoposide, cisplatin), EVAP including multivariate analyses, that (etoposide, vinblastine, doxorubicin, prednisone), or BEACOPP (bleomycin, etoposide, doxorubicin, topoisomerase II␣ is a reliable cyclophosphamide, vincristine, procarbazine, prednisone), topoisomerase II␣ and Ki67 are indicator of tumour aggressiveness. expressed in up to 90% of Hodgkin’s cells. Antibody KiS2, specific for S to M phase, stains only up to 30% of Hodgkin’s cells and few lymphocytes. Antibodies to topoisomerase I (C21; YC Cheng, Expression of the enzyme Yale University) stain all Hodgkin’s cells as well as the majority of surrounding lymphocytes. independently predicted the survival of patients with soft-tissue sarcoma42 specific but reflects malignancy in several ways. For and was the second strongest prognostic factor (after nodal instance, the expression of topoisomerases is commonly status) for both overall and metastasis-free survival in a upregulated in malignant transformed cells, probably cohort of 863 patients with infiltrating breast cancer.43 In a through deficiency in two negative regulators of series of 356 node-negative invasive mammary carcinomas, topoisomerase II expression and activity, p53 and pRB, topoisomerase II␣ and tumour size were the only which are known to be deleted or inactivated in many independent prognostic factors for overall and disease-free human cancers. Furthermore, loss of p53 function is likely survival.44 to impede the detection of DNA damage and to abrogate one important apoptotic pathway, thus allowing the DNA topoisomerase II as a target of survival of genetically degenerate cells. This mechanism chemotherapy may be another way in which the combination of increased Topoisomerase II is such a successful drug target because it topoisomerase II activity with defective p53 function may has an indispensable function in cell biology and it lacks foster genetic instability. Accordingly, accessory aberrations biological redundancy. Inhibitors of this enzyme have are found in association with deregulated topoisomerase become central parts of both primary and adjuvant expression. By analysis of a cohort of 230 patients with chemotherapy regimens in neoplastic diseases, and they breast cancer, Järvinen and colleagues showed that probably will remain so for the foreseeable future.45 topoisomerase II␣ is associated not only with proliferation, Although they form several distinct substance groups, they but also with absence of oestrogen receptors, progesterone are best categorised in two classes according to their receptors, or both, high malignancy grade, DNA biological properties—the topoisomerase poisons and the aneuploidy, and overexpression of HER2/neu catalytic inhibitors. Though as yet unproven, the common belief is that the ␣ isoform is the main pharmacological oncoprotein.37 As an alternative mechanism of topoisomerase IIα target in cancer cells. However, several studies point to an overexpression, amplifications of the coding gene have been additional response of topoisomerase II to cytotoxic reported. These occur, for instance, in association with therapy. amplifications of the HER2/neu gene, in non-small-cell lung carcinoma and breast cancer.38,39 Since the HER2/neu and The topoisomerase II poisons TOP2␣ genes are close to each other on the long arm of Topoisomerase II-poisons are defined as drugs that stabilise chromosome 17, amplification of one gene locus could affect the cleavable complex to cause DNA breaks (figure 1). They
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DNA topoisomerase II in oncology
are called poisons because they convert this essential nuclear enzyme into a lethal poison.9 However, the pathway from DNA breaks to cell death by necrosis or apoptosis is still insufficiently explored.46 During the catalytic cycle of topoisomerase II, gaps in the DNA double strand in the form of cleavable complexes (CC) are present before (CC1; in figure 1 stage 3) and after (CC2; stage 4) DNA processing, exposing a weak point to the attack of intercalating substances. Of these substances, the anthracycline, daunorubicin, stabilises CC1, whereas amsacrine stabilises CC2, and etoposide acts on both CC1 and CC2. These different mechanisms of action nevertheless abut on a common endpoint since they all interfere with the re-ligase activity of topoisomerase II and thus perpetuate the DNA nicks that are thought to be the cause of apoptosis. Such strand breaks are thought to trigger the activity of caspases, which are in charge of programmed cell death.47 However, apoptosis could also be a consequence of aberrant non-homologous recombination after DNA damage48 or oxidative stress induced by topoisomerase poisons.49 Yet another poorly recognised mechanism is the downregulation and caspase-mediated proteolysis of protein kinase C␦ by etoposide targeting of the cytoplasmic fraction of topoisomerase II.50,51 Clearly, therefore, a definite danger lurks behind topoisomerase-directed therapy regimens. Indeed, many tumour cells are defective in apoptotic mechanisms, which may be related to inadequate detection of DNA damage (eg, by mutation of the ATM [ataxia-teleangiectasia-mutated] or p53 genes), deteriorated pathways signalling for cell-cycle arrest or cell death (involving pRB and p53 as higher-order regulators), or overexpression of antiapoptotic proteins such as BCL-2. Topoisomerase II itself may contribute to the disruption of at least one of these mechanisms, since it can break DNA in the region of chromosome 11q23 that includes the ATM gene.52 Such disturbances of normal control mechanisms are likely to allow survival of cells after illegitimate DNA recombination or inaccurate redistribution of daughter chromosomes under the influence of topoisomerase II poisons.36 In this way, not only the selection of more aggressive clones, but also the formation of secondary malignant disorders may be side-effects of a therapy involving topoisomerase poisons. The most frequent therapy-related neoplasias are acute leukaemias.53,54 Hence, neoplastic cells must be killed as efficiently as possible while the minimum injury is inflicted on untransformed cells. This aim can be approached in several ways: by identification of the tumours that would be expected to respond to topoisomerase II-poisons; by reduction of the toxic side-effects through combined regimens; and by more precise selection of the drug target. There is accumulating evidence that the sensitivity of tumour cells to topoisomerase poisons is proportional to their enzyme content.55–57 This idea may seem paradoxical because it implies a diminished drug-to-target ratio. The argument in favour of such a relation is, however, that higher expression of topoisomerase II will result in increased cleavage activity, and that the number of DNA strand breaks arising may be a critical determinant of cell death. Thus, for
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Review instance, coamplification of the HER2/neu and TOP2␣ genes is associated with increased sensitivity to amsacrine39 and anthracyclines,58 whereas deletion of topoisomerase II␣ in HER2-amplified breast tumours induces primary resistance to anthracyclines.58 The assessment of both HER2/neu and topoisomerase II␣ expression might therefore allow selection of patients who would benefit from combined treatment with trastuzumab (Herceptin) and a topoisomerase II␣-poison, whereas little benefit would be expected from the latter if the TOP2␣ gene were deleted. Transfection of heregulin 2 into breast-cancer cells produces a striking increase in their sensitivity to doxorubicin and etoposide.59 An analogous effect was observed when Ewing’s sarcoma cell lines were transfected with the E1A gene, which simultaneously resulted in downregulation of HER2/neu and upregulation of topoisomerase II␣.60 These observations suggest a close relation between the expression of the two genes. A problem in therapeutic designs nevertheless arises from the fact that the tumour aggressiveness associated with high proliferative activity generally over-rides the cell killing achieved by cytotoxic therapy.43 Surprisingly, the expression of topoisomerase II seems to be almost as important as that of the ␣ form for the sensitivity of malignant cells to topoisomerase II inhibitors. Studies on AML blasts from 72 patients revealed no correlation between topoisomerase II␣ activity and sensitivity to topoisomerase II-poisons, although activity of this isoenzyme in the tumour cells could be directly inhibited by incubation with the drugs in vitro. By contrast, topoisomerase II activity, which was not greatly inhibited by these compounds, was inversely correlated with the sensitivity of the cells. These findings were significant for idarubicin (p=0·017) and daunorubicin (p=0·006). Accordingly, resistant cells from patients (IC50 median) had the highest topoisomerase II activity. Cells from patients who relapsed after initial treatment with anthracyclincontaining regimens had a significantly decreased ratio of topoisomerase II␣/ activity than those who did not relapse (p=0·0276).61 The biological background of this intriguing finding nevertheless needs further investigation. Differential targeting of the two isoforms may play an important part in the action of topoisomerase II-poisons. Thus, etoposide,62 mitoxantrone, and amsacrine and its carbamate analogues16 act on both enzymes. Amsacrine seems to inhibit topoisomerase II preferentially,60 and there is evidence that low or absent expression of this isoform may result in very low sensitivity to amsacrine and related compounds.63 Other drugs have been developed that either target both isoenzymes, as chloroquinoxaline sulphonamide does,64 or specifically inhibit topoisomerase II, such as XK469.65 Because the specific functions of topoisomerase II are largely unknown, general application of drugs directed against this isoform may be difficult to envisage. However, the usefulness of such compounds is evident for tumours in which mutant forms of topoisomerase II␣ elude the effect of inhibitors.63 Moreover, the large fraction of G0/G1 cells found in most solid tumours66,67 may be sensitive to topoisomerase
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Review II inhibitors, as shown by the in vitro efficacy of XK469 as a solid-tumour-selective agent.68
DNA topoisomerase II in oncology
effectors of apoptosis. These properties may acquire clinical significance when classic treatment protocols are complicated by drug-resistance syndromes.
The topoisomerase II catalytic inhibitors In contrast to the poisons, the catalytic inhibitors do not induce DNA breaks and may even prevent their formation. They act at any stage in the enzymatic cycle other than the cleavable complex (figure 1), either by interference with topoisomerase II binding to DNA, or by locking the DNAbound enzyme in the shape of a closed clamp. Hence, two new therapeutic approaches based on the combination of a poison with a catalytic inhibitor in cancer treatment have been designed. Pharmacological modulation of a poison by a catalytic inhibitor
Since a catalytic inhibitor can abolish the toxic effect of a poison, this type of inhibitor can be used to direct the toxic effect. For example, the hydrophilic bisdioxopiperazine dexrazoxane (ICRF-187), which does not cross the bloodbrain barrier, can be used to increase the tolerated dose of etoposide, which does.69 This principle is currently being studied in a phase I/II trial in patients with metastases to the central nervous system from small-cell lung cancer. Another indication of counteracting a topoisomerase II poison with a catalytic inhibitor is given in the treatment of accidental anthracycline extravasation, in which the severe tissue necrosis can be significantly attenuated by use of dexrazoxane.70 A potentially much broader use of combining a protective catalytic inhibitor with a poison is suggested by the physiological propensity of most solid tumours to have acid extracellular environments. Weak bases cannot permeate the plasma membrane, but the neutral drug etoposide can; therefore, a non-toxic weakly alkaline catalytic inhibitor would be able to protect the normal tissues in the body without affecting the antitumour efficacy of etoposide. This idea has been successfully demonstrated in preclinical work with the weak base chloroquine, a malarial drug that is also a topoisomerase II catalytic inhibitor.71 However, chloroquine is too toxic for in vivo use, so new agents with the desired properties will have to be developed. Sequential use of a catalytic inhibitor with a poison
The lack of cross-resistance to catalytic inhibitors such as bisdioxopiperazines in cell lines selected for resistance to topoisomerase II-poisons makes the use of bisdioxopiperazines in the second-line treatment of malignant tumours a promising idea. In Japan, sobuzoxane (MST-16) has been approved for clinical use, which emphasises the pharmacological potential of the bisdioxopiperazine family in clinical settings. There are nevertheless exceptions to the suggestion of an alternative use of topoisomerase II catalytic inhibitors in tumour therapy. Thus, experimental compounds dexrazoxane and merbarone can induce apoptosis in the absence of DNA strand breaks.72 In both instances, this process seems to be related to the activation of caspase-like and ICE/CED3-like proteases, which are known to be distal
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Drug resistance The emperor of Pont, Mithridate VII (120–63 BC), intended to escape assassination by accustoming his metabolism to several popular poisons. By habitual intake of slowly increasing doses, he managed to become insensitive to these drugs. He was nevertheless successfully killed by administration of a substance he had neglected. This principle of developing and circumventing drug resistance is similar to that used today in experimental research and clinical therapy. Indeed, cells grown in the presence of increasing concentrations of cytotoxic drugs may become refractory not only to the substance applied, but also to structurally unrelated compounds, acquiring the multidrugresistance phenotype. Multidrug resistance to topoisomerase II poisons exists in two major forms: one is attributable to efflux pumps in the cell membrane that lower the steady-state concentration of the drug at its target site (transport or target multidrug resistance); in the other form, the activity and sensitivity of the target enzyme topoisomerase II itself are decreased by downregulation or mutation (altered topoisomerase II multidrug resistance). The classic multidrug-resistance phenotype, whereby a cell becomes resistant not only to the selecting drug but also to structurally unrelated drugs, has stimulated a tremendous amount of research activity, which has been reviewed elsewhere and is beyond the scope of this review.73,74 The altered topoisomerase II multidrug-resistance phenotype, associated with a reduction in topoisomerase II content and activity, was first described in 1986 and 1988.75,76 That this phenotype could also be caused by a mutation in topoisomerase II was discovered by Hinds and colleagues in 1991.77 Subsequently, other mutations in human topoisomerase II were shown to be functional in a transfected yeast system.78 The mutations inducing resistance to topoisomerase II poisons cluster around the Walker B consensus ATP-binding site or around the Y805 catalytic tyrosine,79 whereas mutations causing resistance to the bisdioxopiperazine type catalytic inhibitors cluster in and around the Walker A ATP-binding site.80 These observations further attest to the fundamental difference between these two classes of compounds. From a clinical perspective, the significance of the multidrug-resistance phenotype is difficult to assess. Studies on the expression of drug pumps, which are mostly done by immunohistochemistry, in many, but by no means all, cases detect a correlation between overexpression of a pump and clinical resistance. P-glycoprotein expression has been studied to such an extent that two meta-analyses have been done, one on AML81 and one on breast cancer,82 both documenting for this pumps relevance in clinical drug resistance. The altered topoisomerase II multidrugresistance phenotype has been much less studied because changes in topoisomerase II expression in clinical material have long been difficult to measure. However, mutations in
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Search strategy and selection criteria Published data for this review were searched by using Current Contents and PubMed; historical data (before the 1960s) were found by secondary citation. Articles were chosen by their relevance and only those published in English were selected. We also contacted researchers to obtain unpublished data.
topoisomerase II do not seem to have a major role in resistance; the few studies so far found no mutations83,84 or a low frequency (eg, one in 13 cases85). The H69/VP etoposide-resistant cell line, which overexpresses both P-glycoprotein and multidrugresistance-associated protein, another transmembrane pump, was eventually shown to have the altered topoisomerase II multidrug-resistance phenotype, with a splice-site mutation in topoisomerase II.86 Moreover, this H69/VP strain shows moderate overexpression of lung resistance protein, but not of breast-cancer resistance protein or mitoxantrone-resistance protein (unpublished). That a single drug-selected resistant cell line can simultaneously display four of the five known resistance phenotypes highlights the complexity that bedevils the clinical application of the information on multidrug resistance collected through decades of laboratory research. However, this experience may be helpful in selection of new agents destined to circumvent the various multidrugresistance phenotypes by being poor substrates for the efflux pumps, or like topoisomerase II catalytic inhibitors, by attacking the target in a different way.
Conclusions Topoisomerase II-targeting drugs have been used extensively in cancer chemotherapy since the clinical introduction of daunorubicin in 1964, and will continue to be used for the foreseeable future. Although the new cytostatic drug classes, such as those that inhibit signal transduction, telomerase, angiogenesis, or proteases, will be of increasing real benefit to cancer patients, current preclinical and clinical studies show that cytostatic agents generally have to be combined with cytotoxic drugs to achieve their full efficacy. The vast amount of knowledge and experience gained over the past four decades in the use of topoisomerase II anticancer agents should aid in developing even better ways of attacking this specific drug target. Acknowledgments
We thank Bernd Wüsthoff and Angela Kellner for helpful discussions during the preparation of this paper. References
1 Berger JM, Gamblin SJ, Harrison SC, Wang JC. Structure and mechanism of DNA topoisomerase II. Nature 1996; 379: 225–32. 2 Wang JC. DNA topoisomerases: new break for archaeal enzyme. Nature 1997; 386: 329–31. 3 Slevin ML. The clinical pharmacology of etoposide. Cancer 1991; 67: 319–29. 4 Tewey KM, Rowe TC, Yang L, et al. Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. Science 1984; 226: 466–68.
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Review 5 Nitiss JL. Investigating the biological functions of DNA topoisomerases in eukaryotic cells. Biochim Biophys Acta 1998; 1400: 63–81. 6 Tsai-Pflugfelder M, Liu LF, Liu AA, et al. Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21–22. Proc Natl Acad Sci USA 1988; 85: 7177–81. 7 Jenkins JR, Ayton P, Jones T, et al. Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24. Nucleic Acids Res 1992; 20: 5587–92. 8 Biersack H, Jensen S, Gromova I, et al. Active heterodimers are formed from human DNA topoisomerase II ␣ and II  isoforms. Proc Natl Acad Sci USA 1996; 93: 8288–93. 9 Wang JC. DNA topoisomerases. Annu Rev Biochem 1996; 65: 635–92. 10 Stevnsner T, Bohr VA. Studies on the role of topoisomerases in general, gene- and strand- specific DNA repair. Carcinogenesis 1993; 14: 1841–50. 11 Rudolph P, Tronnier M, Menzel R, et al. Enhanced expression of Ki-67, topoisomerase II␣, PCNA, p53 and p21WAF1/Cip1 reflecting proliferation and repair activity in UV-irradiated melanocytic nevi. Hum Pathol 1998; 29: 1480–87. 12 Isaacs RJ, Davies SL, Sandri MI, et al. Physiological regulation of eukaryotic topoisomerase II. Biochim Biophys Acta 1998; 1400: 121–37. 13 Woessner RD, Mattern MR, Mirabelli CK, et al. Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells. Cell Growth Differ 1991; 2: 209–14. 14 Adachi N, Nomoto M, Kohno K, Koyama H. Cell-cycle regulation of the DNA topoisomerase II␣ promoter is mediated by proximal CCAAT boxes: possible involvement of acetylation. Gene 2000; 245: 49–57. 15 Marsh KL, Willmore E, Tinelli S, et al. Amsacrine-promoted DNA cleavage site determinants for the two human DNA topoisomerase II isoforms alpha and beta. Biochem Pharmacol 1996; 52: 1675–85. 16 Errington F, Willmore E, Tilby MJ, et al. Murine transgenic cells lacking DNA topoisomerase II are resistant to acridines and mitoxantrone: analysis of cytotoxicity and cleavable complex formation. Mol Pharmacol 1999; 56: 1309–16. 17 Yang X, Li W, Prescott ED, et al. DNA topoisomerase II and neural development. Science 2000; 287: 131–34. 18 Sng JH, Heaton VJ, Bell M, et al. Molecular cloning and characterization of the human topoisomerase II␣ and II genes: evidence for isoform evolution through gene duplication. Biochim Biophys Acta 1999; 1444: 395–406. 19 Shapiro PS, Whalen AM, Tolwinski NS, et al. Extracellular signalregulated kinase activates topoisomerase II␣ through a mechanism independent of phosphorylation. Mol Cell Biol 1999; 19: 3551–60. 20 Kellner U, Heidebrecht HJ, Rudolph P, et al. Detection of human topoisomerase II alpha in cell lines and tissues: characterization of five novel monoclonal antibodies. J Histochem Cytochem 1997; 45: 251–63. 21 Escargueil AE, Plisov SY, Skladanowski A, et al. Recruitment of cdc2 kinase by DNA topoisomerase II is coupled to chromatin remodeling. FASEB J 2001; 15: 2288–90. 22 Wang Q, Zambetti GP, Suttle DP. Inhibition of DNA topoisomerase II␣ gene expression by the p53 tumor suppressor. Mol Cell Biol 1997; 17: 389–97. 23 Cowell IG, Okorokov AL, Cutts SA, et al. Human topoisomerase II␣ and II interact with the C-terminal region of p53. Exp Cell Res 2000; 255: 86–94. 24 Kingma PS, Osheroff N. The response of eukaryotic topoisomerases to DNA damage. Biochim Biophys Acta 1998; 1400: 223–32. 25 Carrier F, Georgel PT, Pourquier P, et al. Gadd45, a p53-responsive stress protein, modifies DNA accessibility on damaged chromatin. Mol Cell Biol 1999; 19: 1673–85. 26 Hochhauser D, Valkov NI, Gump JL, et al. Effects of wild-type p53 expression on the quantity and activity of topoisomerase II␣ and  in various human cancer cell lines. J Cell Biochem 1999; 75: 245–57. 27 Bhat UG, Raychaudhuri P, Beck WT. Functional interaction between human topoisomerase II␣ and retinoblastoma protein. Proc Natl Acad Sci USA 1999; 96: 7859–64. 28 Parwaresch R, Rudolph P. The cell cycle: theory and applications to cancer. Onkologie 1996; 19: 464–72. 29 van Hemert MJ, Steensma HY, van Heusden GP. 14-3-3 proteins:
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key regulators of cell division, signalling and apoptosis. Bioessays 2001; 23: 936–46. Kurz EU, Leader KB, Kroll DJ, et al. Modulation of human DNA topoisomerase II␣ function by interaction with 14-3-3⑀. J Biol Chem 2000; 275: 13948–54. Razin SV. Chromosomal DNA loops may constitute basic units of the eukaryotic genome organization and evolution. Crit Rev Eukaryot Gene Expr 1999; 9: 279–83. Charron M, Hancock R. Chromosome recombination and defective genome segregation induced in Chinese hamster cells by the topoisomerase II inhibitor VM-26. Chromosoma 1991; 100: 97–102. Pedersen-Bjergaard J, Brondum-Nielsen K, Karle H, Johansson B. Chemotherapy-related, late-occurring, Philadelphia chromosome in AML, ALL and CML: similar events related to treatment with DNA topoisomerase II inhibitors? Leukemia 1997; 11: 1571–74. Huai Q, Colandene JD, Chen Y, et al. Crystal structure of NaeI: an evolutionary bridge between DNA endonuclease and topoisomerase. EMBO J 2000; 19: 3110–18. McClintock B. Chromosome organization and gene expression. Cold Spring Harbor Symp Quant Biol 1951; 16: 13–47. Baguley BC, Ferguson LR. Mutagenic properties of topoisomerasetargeted drugs. Biochim Biophys Acta 1998; 1400: 213–22. Järvinen TA, Kononen J, Pelto-Huikko M, Isola J. Expression of topoisomerase II␣ is associated with rapid cell proliferation, aneuploidy, and c-erbB2 overexpression in breast cancer. Am J Pathol 1996; 148: 2073–82. Keith WN, Tan KB, Brown R. Amplification of the topoisomerase II ␣ gene in a non-small cell lung cancer cell line and characterisation of polymorphisms at the human topoisomerase II␣ and  loci in normal tissue. Genes Chromosomes Cancer 1992; 4: 169–75. Smith K, Houlbrook S, Greenall M, et al. Topoisomerase II ␣ co-amplification with erbB2 in human primary breast cancer and breast cancer cell lines: relationship to m-AMSA and mitoxantrone sensitivity. Oncogene 1993; 8: 933–38. Sampson SA, Kreipe H, Gillett CE, et al. KiS1–a novel monoclonal antibody which recognizes proliferating cells: evaluation of its relationship to prognosis in mammary carcinoma. J Pathol 1992; 168: 179–85. Rudolph P, Lappe T, Schubert C, et al. Diagnostic assessment of two novel proliferation-specific antigens in benign and malignant melanocytic lesions. Am J Pathol 1995; 147: 1615–25. Rudolph P, Kellner U, Chassevent A, et al. Prognostic relevance of a novel proliferation marker, Ki-S11, for soft-tissue sarcoma: a multivariate study. Am J Pathol 1997; 150: 1997–2007. Rudolph P, MacGrogan G, Bonchion F, et al. Prognostic significance of ki-67 and topoisomerase II␣ expression in infiltrating ductal carcinoma of the breast. Breast Cancer Res Treat 1999; 55: 61–71. Rudolph P, Olsson H, Bonatz G, et al. Correlation between p53, c-erbB-2, and topoisomerase II␣ expression, DNA ploidy, hormonal receptor status and proliferation in 356 node-negative breast carcinomas: prognostic implications. J Pathol 1999; 187: 207–16. Hande KR. Clinical applications of anticancer drugs targeted to topoisomerase II. Biochim Biophys Acta 1998; 1400: 173–84. Kaufmann SH. Cell death induced by topoisomerase-targeted drugs: more questions than answers. Biochim Biophys Acta 1998; 1400: 195–211. Benjamin CW, Hiebsch RR, Jones DA. Caspase activation in MCF7 cells responding to etoposide treatment. Mol Pharmacol 1998; 53: 446–50. Cummings J, Smyth JF. DNA topoisomerase I and II as targets for rational design of new anticancer drugs. Ann Oncol 1993; 4: 533–43. Taatjes DJ, Fenick DJ, Gaudiano G, Koch TH. A redox pathway leading to the alkylation of nucleic acids by doxorubicin and related anthracyclines: application to the design of antitumor drugs for resistant cancer. Curr Pharm Des 1998; 4: 203–18. Dal Pra I, Whitfield JF, Chiarini A, Armato U. Changes in nuclear protein kinase C-delta holoenzyme, its catalytic fragments, and its activity in polyomavirus-transformed pyF111 rat fibroblasts while proliferating and following exposure to apoptogenic topoisomerase II inhibitors. Exp Cell Res 1999; 249: 147–60. Lothstein L, Israel M, Sweatman TW. Anthracycline drug targeting: cytoplasmic versus nuclear–a fork in the road. Drug Resist Update 2001; 4: 169–77. Kaufmann WK. Human topoisomerase II function, tyrosine
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phosphorylation and cell cycle checkpoints. Proc Soc Exp Biol Med 1998; 217: 327–34. Andersen MK, Christiansen DH, Jensen BA, et al. Therapy-related acute lymphoblastic leukaemia with MLL rearrangements following DNA topoisomerase II inhibitors, an increasing problem: report on two new cases and review of the literature since 1992. Br J Haematol 2001; 114: 539–43. Seiter K, Feldman EJ, Sreekantaiah C, et al. Secondary acute myelogenous leukemia and myelodysplasia without abnormalities of chromosome 11q23 following treatment of acute leukemia with topoisomerase II-based chemotherapy. Leukemia 2001; 15: 963–70. Houlbrook S, Harris AL, Carmichael J, Stratford IJ. Relationship between topoisomerase II levels and resistance to topoisomerase II inhibitors in lung cancer cell lines. Anticancer Res 1996; 16: 1603–10. MacGrogan G, Mauriac L, Durand M, et al. Immunoexpression of DNA topoisomerase II␣ predicts response to primary chemotherapy in breast cancer patients. Proc Am Assoc Cancer Res 2001; 42: (Abstr) 4596 Koshiyama M, Fujii H, Kinezaki M, et al. Immunohistochemical expression of topoisomerase IIalpha (Topo II␣) and multidrug resistance-associated protein (MRP), plus chemosensitivity testing, as chemotherapeutic indices of ovarian and endometrial carcinomas. Anticancer 2001; 21: 2925–32. Järvinen TA, Tanner M, Rantanen V, et al. Amplification and deletion of topoisomerase II␣ associate with ErbB-2 amplification and affect sensitivity to topoisomerase II inhibitor doxorubicin in breast cancer. Am J Pathol 2000; 156: 839–47. Harris LN, Yang L, Tang C, et al. Induction of sensitivity to doxorubicin and etoposide by transfection of MCF-7 breast cancer cells with heregulin beta-2. Clin Cancer Res 1998; 4: 1005–12. Zhou Z, Zwelling LA, Ganapathi R, Kleinerman ES. Enhanced etoposide sensitivity following adenovirus-mediated human topoisomerase IIalpha gene transfer is independent of topoisomerase IIbeta. Br J Cancer 2001; 85: 747–51. Gieseler F, Glasmacher A, Kampfe D, et al. Topoisomerase II activities in AML and their correlation with cellular sensitivity to anthracyclines and epipodophyllotoxines. Leukemia 1996; 10: 1177–80. Willmore E, Frank AJ, Padget K, et al. Etoposide targets topoisomerase II␣ and II in leukemic cells: isoform-specific cleavable complexes visualized and quantified in situ by a novel immunofluorescence technique. Mol Pharmacol 1998; 54: 78–85. Herzog CE, Holmes KA, Tuschong LM, et al. Absence of topoisomerase II in an amsacrine-resistant human leukemia cell line with mutant topoisomerase II␣. Cancer Res 1998; 58: 5298–300. Gao H, Yamasaki EF, Chan KK, et al. Chloroquinoxaline sulfonamide (NSC 339004) is a topoisomerase II␣/ poison. Cancer Res 2000; 60: 5937–40. Gao H, Huang KC, Yamasaki EF, et al. XK469, a selective topoisomerase II poison. Proc Natl Acad Sci USA 1999; 96: 12168–73. Rudolph P, Knuchel R, Endl E, et al. The immunohistochemical marker Ki-S2: cell cycle kinetics and tissue distribution of a novel proliferation-specific antigen. Mod Pathol 1998; 11: 450–56. Rudolph P, Alm P, Olsson H, et al. Concurrent overexpression of p53 and c-erbB-2 correlates with accelerated cycling and concomitant poor prognosis in node-negative breast cancer. Hum Pathol 2001; 32: 311–19. Corbett TH, LoRusso P, Demchick L, et al. Preclinical antitumor efficacy of analogs of XK469: sodium-(2-[4-(7-chloro-2quinoxalinyloxy)phenoxy]propionate. Invest New Drugs 1998; 16: 129–39. Holm B, Jensen PB, Sehested M. ICRF-187 rescue in etoposide treatment in vivo: a model targeting high-dose topoisomerase II poisons to CNS tumors. Cancer Chemother Pharmacol 1996; 38: 203–09. Langer SW, Sehested M, Jensen PB. Treatment of anthracycline extravasation with dexrazoxane. Clin Cancer Res 2000; 6: 3680–86. Jensen PB, Sorensen BS, Sehested M, et al. Targeting the cytotoxicity of topoisomerase II-directed epipodophyllotoxins to tumor cells in acidic environments. Cancer Res 1994; 54: 2959–63. Khelifa T, Beck WT. Merbarone, a catalytic inhibitor of DNA topoisomerase II, induces apoptosis in CEM cells through activation of ICE/CED-3-like protease. Mol Pharmacol 1999; 55: 548–56. Volm M. Multidrug resistance and its reversal. Anticancer Res 1998; 18: 2905–17.
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74 Lehne G. P-glycoprotein as a drug target in the treatment of multidrug resistant cancer. Curr Drug Targets 2000; 1: 85–99. 75 Pommier Y, Kerrigan D, Schwartz RE, et al. Altered DNA topoisomerase II activity in Chinese hamster cells resistant to topoisomerase II inhibitors. Cancer Res 1986; 46: 3075–81. 76 Danks MK, Schmidt CA, Cirtain MC, et al. Altered catalytic activity of and DNA cleavage by DNA topoisomerase II from human leukemic cells selected for resistance to VM-26. Biochemistry 1988; 27: 8861–69. 77 Hinds M, Deisseroth K, Mayes J, et al. Identification of a point mutation in the topoisomerase II gene from a human leukemia cell line containing an amsacrine-resistant form of topoisomerase II. Cancer Res 1991; 51: 4729–31. 78 Hsiung Y, Jannatipour M, Rose A, et al. Functional expression of human topoisomerase II␣ in yeast: mutations at amino acids 450 or 803 of topoisomerase II␣ result in enzymes that can confer resistance to anti-topoisomerase II agents. Cancer Res 1996; 56: 91–99. 79 Vassetzky YS, Alghisi GC, Gasser SM. DNA topoisomerase II mutations and resistance to anti-tumor drugs. Bioessays 1995; 17: 767–74. 80 Freire R, d’Adda DF, Wu L, et al. Cleavage of the Bloom’s syndrome gene product during apoptosis by caspase-3 results in an impaired
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interaction with topoisomerase III␣. Nucleic Acids Res 2001; 29: 3172–80. Bowman KJ, Newell DR, Calvert AH, Curtin NJ. Differential effects of the poly (ADP-ribose) polymerase (PARP) inhibitor NU1025 on topoisomerase I and II inhibitor cytotoxicity in L1210 cells in vitro. Br J Cancer 2001; 84: 106–12. Bailly C, Laine W, Baldeyrou B, et al. DNA intercalation, topoisomerase II inhibition and cytotoxic activity of the plant alkaloid neocryptolepine. Anticancer Drug Des 2000; 15: 191–201. Danks MK, Warmoth MR, Friche E, et al. Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells. Cancer Res 1993; 53: 1373–79. Kaufmann SH, Karp JE, Jones RJ, et al. Topoisomerase II levels and drug sensitivity in adult acute myelogenous leukemia. Blood 1994; 83: 517–30. Kubo A, Yoshikawa A, Hirashima T, et al. Point mutations of the topoisomerase II␣ gene in patients with small cell lung cancer treated with etoposide. Cancer Res 1996; 56: 1232–36. Wessel I, Jensen PB, Falck J, et al. Loss of amino acids 1490Lys-SerLys1492 in the COOH-terminal region of topoisomerase II␣ in human small cell lung cancer cells selected for resistance to etoposide results in an extranuclear enzyme localization. Cancer Res 1997; 57: 4451–54.
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Culprit and victim – DNA topoisomerase II Udo Kellner, Maxwell Sehested, Peter B Jensen, Frank Gieseler, and Pierre Rudolph
The phylogenetic antiquity of DNA topoisomerases indicates their vital function. Structure and maintenance of genomic DNA depend on the activity of these enzymes, and without them DNA replication and cell division are impossible. Topoisomerase II␣ has therefore become the main target of many antitumour therapy regimens, even though the exact mechanism of cell killing remains elusive. The success of this approach is limited by the development of spontaneous resistance, and druginduced DNA damage can increase malignancy. Nevertheless, the combined use of topoisomeraseinhibiting drugs with different mechanisms of action promises to improve particular treatment designs. The degree of topoisomerase II expression in tumours may predict the clinical course and responsiveness to therapy. Lancet Oncol 2002; 3: 235–43
Topoisomerases are enzymes that can modify (isomerise) the tertiary structure of DNA without changing its primary structure,1 which is determined by the nucleotide sequence. The high degree of conservation of these enzymes among prokaryotes and eukaryotes2 indicates an essential role in cell biology. In human beings, two classes of topoisomerases are well characterised—type I and type II, of which the latter will be discussed in this review. All physiological functions of DNA depend on its tertiary configuration, and therefore the activity of topoisomerase is of vital importance for all DNA-containing cells (figure 1). Because its structure is a double helix, DNA is under tortional stress that results in multiplex twisting of the molecule. To be processed for replication or gene expression, the supercoiled DNA must become accessible to polymerases or components of the transcription machinery. This change requires relaxation and untangling of the intertwined DNA strands, which are the typical tasks of DNA topoisomerases. The anthracyclines and epipodophyllotoxins have been mainstays in medical oncology since the 1960s, and were eventually shown to target DNA topoisomerase II. Poisons of the enzyme and related compounds were among the earliest drugs used in cancer therapy; the first written mention of podophyllotoxins in cancer treatment dates from the 8th century.3 Efforts made during the 1950s to increase the efficacy of the native substances by minor chemical modifications led to the development of the first two semisynthetic (epi-)podophyllotoxin derivatives, etoposide and teniposide, which unlike the parent podophyllotoxins do not inhibit microtubule assembly but do inhibit topoisomerase II. Clinical trials started in 1971 and showed activity in acute myeloid leukaemia (AML), Hodgkin’s disease, non-Hodgkin lymphoma, and
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ATPase
T
ATP
B 2
A
G
1 3
ATP+Pi
(CC1)
5 4 (CC2)
3'
Figure 1. Proposed structure and catalytic cycle of topoisomerase II. One DNA strand ‘G’ (gate) is cleaved, allowing the passage of another intact DNA strand ‘T’ (transport).The enzyme is a homodimer consisting of three functional segments, an ATPase (top), a cleavage (B), and a C-terminal (A) part. The topoisomerase-II poisons act by stabilising stage 3 or 4, where the DNA strand is cleaved. The catalytic inhibitors act either at stage 1 (chloroquine and aclarubicin) or at stage 5 (bisdioxopiperazines such as dexrazoxane). Figure modified from reference 1.
carcinomas of the lung, stomach, breast, and ovaries. Etoposide is now a standard agent in treatment of small-cell lung cancer and testicular cancer (table 1). The antibiotic group of anthracyclines, which are fermentation products of Streptomyces peuceus, entered clinical oncology in the 1960s. However, these antibiotics were not recognised to be directed against topoisomerase II until 1984.4 Anthracyclines are now widely used, the most commonly is doxorubicin which can be used for for lymphomas, breast cancer, sarcomas, Kaposi’s sarcoma, and leukaemias. Epirubicin, which may be less cardiotoxic than doxorubicin, is extensively used in Europe for treatment of breast cancer, and it has recently been approved by the US Food and Drug Administration for adjuvant therapy of breast cancer (table 1). Daunorubicin and idarubicin are used in acute leukaemias. Other clinically applied topoisomerase II-poisons are mitoxantrone, an anthracenedione for acute leukaemias, breast and ovarian cancers, bladder cancer, and lymphomas, and amsacrine in acute leukaemia. UK is at the Department of Pathology, University of Magdeburg, Germany. MS and PBJ are at the Finsen and Laboratory Centres, Rigshospitalet, Copenhagen, Denmark. FG is at the Section of Hematology/Oncology, Department of Internal Medicine, University Hospital of Kiel, Germany. PR is at the Department of Pathology, University of Kiel, Germany. Correspondence: Dr Udo Kellner, Institut für Pathologie, Otto-von-Guericke Universität, 39120 Magdeburg, Germany. Tel: +49 391 67 14 295. Fax: +49 391 67 190 646. Email: [email protected]
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Table 1. Overview of the topoisomerase-II-targeting drugs in current clinical use and the range of successful applications Drug
Class
Applications
Topoisomerase-II␣ poisons Epirubicin
Anthracycline
Adjuvant breast cancer
Doxorubicin
Anthracycline
Idarubicin
Anthracycline
Non-Hodgkin lymphoma, chronic lymphocytic leukaemia, Hodgkin’s disease, multiple myeloma, acute leukaemia, small-cell lung cancer, breast, ovarian, and bladder cancers, sarcomas Acute leukaemia
Daunorubicin
Anthracycline
Acute leukaemia
Mitoxantrone
Antracendione
Acute leukaemia, breast, ovarian, and bladder cancers
Amsacrine
Ansidide
Acute leukaemia
Etoposide
Epipodophyllotoxin
Small-cell lung cancer, testicular cancer, ovarian tumours, acute myeloid leukaemia, non-Hodgkin lymphoma
Teniposide
Epipodophyllotoxin
Small-cell lung cancer
Topoisomerase-II␣ catalytic inhibitors Aclarubicin
Anthracycline
Acute myeloid leukaemia
Sobuzoxane
Bisdioxopiperazine
T-cell lymphomas and leukaemia
Dexrazoxane
Bisdioxopiperazine
Central nervous system metastases, anthracycline extravasation
Several resistance mechanisms limit the clinical success of topoisomerase-targeting drugs. Moreover, secondary malignant disorders can arise after topoisomerase-directed treatment regimens. To understand the benefits and disadvantages of these therapeutic approaches, we will take a closer look at the mechanisms of action of topoisomerase II, its role in neoplasia, and the biology of pharmacological topoisomerase inhibition.
Mechanism of action, regulation, and physiological functions Topoisomerase II is the only enzyme able to cleave and religate double-stranded DNA. This enzyme acts in the relaxation of DNA supercoils that accumulate during gene transcription and along with the progression of the replication fork. Moreover, only topoisomerase II can carry out the decatenation of replicated circular double-stranded DNA, and it is obligatorily involved in the remodelling of chromatin during mitosis.5 There are two highly homologous isoforms of topoisomerase II in human beings, which are encoded by different genes. The gene for topoisomerase II␣ is on chromosome 17q21–22 and encodes a 170 kDa protein.6 The topoisomerase II gene, encoding a 180 kDa protein, is on chromosome 3q24.7 The function of topoisomerase II requires dimerisation, mainly in form of homodimers, but heterodimers occur.8 Both enzymes are ATP-dependent endonucleases and ligases that cut both DNA strands and covalently bind to the new DNA 5⬘ end. The catalytic action starts with an energyindependent endonucleic DNA cut and a 5⬘ covalent linking (figure 1). The double strand break is fixed by disulphide bonds bridging the topoisomerase II dimer,1 which constitutes the so-called cleavable complex. Under ATP hydrolysis, the nicked DNA double strand is (re)ligated, and the topoisomerase II dimer eventually dissociates from the DNA. This mode of action explains why topoisomerase II activity is needed at several points in DNA metabolism, such as transcription, replication, and chromosome condensation and segregation.9 In addition, a role for topoisomerase II in nucleotide excision repair has been discussed.10,11
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Several complex mechanisms modulate the activity of topoisomerase II, including transcriptional and posttranscriptional regulation, and post-translational modifications such as phosphorylation, protein interactions, and proteasome-mediated degradation.12 The expression of topoisomerase II␣ is regulated with the cell cycle, whereas that of the II form is not.13 In proliferating cells, topoisomerase II␣ becomes detectable in late G1 phase, and the amount gradually increases to peak in G2/M (figures 2 and 3). The induction of quiescence or terminal differentiation, by contrast, results in striking downregulation of expression of this enzyme. The main transcription factor that brings about transactivation of topoisomerase II␣, nuclear factor Y, binds to the proximal inverted CCAAT boxes in the topoisomerase II␣ promoter region with constant avidity throughout the cell cycle.14 This apparent paradox is explained by the observation that the association of nuclear factor Y with histone acetyltransferase greatly increases topoisomerase II␣ promoter activity, downregulation during G0/G1 being achieved by trichostatin-mediated histone deacetylation.14 The dependency on the cell-cycle phase of topoisomerase II␣ expression supports the concept that the enzyme has a major role in cell proliferation, but there is no compelling evidence indicating involvement of the  isoform. The physiological functions of the latter form are poorly understood, although the two enzymes share the same mechanism of action and target similar cleavage sites.15 Transgenic cell lines lacking topoisomerase II apparently remain viable.16 By contrast, TOP2 knock-out mice die shortly after birth from respiratory insufficiency despite initially normal development during the embryonic period; motor axons fail to contact skeletal muscles.17 Although this finding does not elucidate the physiological role of topoisomerase II, it points to a vital function that is apparently unrelated to cell proliferation. The  isoenzyme may rescue the function of topoisomerase II␣ in proliferating cells when the latter is expressed at inappropriately low concentrations, mutated, or inhibited pharmacologically. This idea is supported by the ability of
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topoisomerase II to form heterodimers, and by evidence suggesting an evolution of the two isoforms through gene duplication.18 Beyond gene expression, phosphorylation of topoisomerase II␣ is required to increase its activity. The main factor involved in this process is casein kinase II, although recent evidence indicates involvement of the mitogen-activated protein kinase extracellular signal-regulated kinase pathway,19 in accordance with an induction of topoisomerase activity in response to mitogenic signals.20 Despite a direct interaction, the mitotic CDC2 kinase (CDK1), which forms together with cyclin B the mitosis-promoting factor MPF, apparently does not phosphorylate Figure 2. Immunohistochemical staining of a small-cell carcinoma of the lung with KiS5. In this topoisomerase II␣ while stimulating tumour type, which can be successfully treated with etoposide and teniposide, topoisomerase II␣ is its catalytic activity and its binding to expressed in up to 70% of the cells. Only cycling cells (G through mitosis) are labelled, with variable intensity reflecting differing expression. The staining pattern is similar to that obtained with the chromatin.21 This newly recognised proliferation marker Ki67. mechanism points to cooperation of the two proteins in chromatin remodelling and explains the breaks.30 This effect, which seemingly is not brought about requirement for topoisomerase II␣ in chromosome by other 14-3-3 proteins, seems to be attributable to a decrease in the DNA-binding activity of topoisomerase II␣, condensation. Activity and expression of topoisomerase II are further suggesting an additional mechanism of physiological influenced by interaction with two master regulators of the regulation. cell cycle, p53, and the retinoblastoma susceptibility gene product, pRB. The stress-activated tumour suppressor Topoisomerase II and tumour biology protein p53 can downregulate topoisomerase II␣ expression In view of the many roles of topoisomerase II in DNA by transcriptional regulation22 and modulate the activity of processing, an oncogenic effect associated with the activity of both the ␣ and  isoenzymes by direct protein interaction.23 these enzymes might be expected. Defective function of This mechanism is probably designed to halt replication in topoisomerase II engendering inappropriate DNA breaks the case of DNA damage. p53 nevertheless seems to exert a can lead to incorrect strand recombination, and dual function, because DNA damage provoked by various chromosomal translocations can result from incorrect agents such as ionising radiation (with the exception of segregation during mitosis. Rearrangements of the genome ultraviolet photoproducts) greatly stimulates the cleavage commonly occur by way of illegitimate recombination activity of topoisomerase II,24 which is mediated by the p53- between DNA loop anchorage sites, and topoisomerase II dependent stress-induced Gadd45 protein.25 Induction of has been implicated in this process.31 Although DNA doublewild-type p53 also seems to result in a large increase in strand breaks are likely to induce a lethal phenotype topoisomerase II content in some cell types.26 The resulting in a mitotic catastrophe in most instances, some biological significance of this process is not clear. In addition cells may survive and eventually acquire genomic to p53, pRB physically interacts with topoisomerase II␣ and aberrations such as chromosomal translocations or DNA efficiently inhibits its activity,27 which discloses another losses that promote malignancy.32,33 Topoisomerase II might conceivably function as a feature of the pRB-mediated negative cell cycle regulation at recombinase, as topoisomerase I does, since substitution of a the G1/S boundary.28 Another interesting facet of topoisomerase II␣ has lately single aminoacid in the restriction endonuclease, NaeI, is emerged—its interaction with a protein from the 14-3-3 enough to elicit both topoisomerase and recombinase family. Although their exact function is largely unknown, activity.34 More probably, however, strand breaks generated mounting evidence suggests that these proteins are involved by unscheduled action of topoisomerases may trigger in several important biological processes such as signal recombination pathways as the cells attempt to repair the transduction, cell-cycle control, apoptosis, and stress damage, or recombinations may arise from spontaneous endresponse in that they act as adaptor molecules stimulating to-end fusion of unprotected DNA strands.35 As a corollary, protein-protein interactions, regulate the subcellular the essentially cytotoxic effects of topoisomerase poisons can location of proteins, and activate or inhibit enzymes.29 We be mutagenic when cells survive the genomic damage.36 Increased expression of topoisomerase II, a common observed that 14-3-3⑀ negatively affects the capacity of etoposide to produce topoisomerase-mediated DNA strand occurrence in neoplasia (figures 2 and 3), is by no means 1
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the other, which would explain the concurrent overexpression of the two gene products in breast cancer.37 Topoisomerase II␣ is a reliable proliferation marker, because the cellular content of the enzyme directly mirrors the proliferative activity of the cells. In 1992, Sampson and coworkers described a monoclonal antibody with affinity for a nuclear antigen in proliferating cells, Ki-S1, which seemed to provide useful prognostic information in breast carcinoma.40 This marker was also found to be a good indicator of malignancy in melanocytic lesions.41 Eventually, the antigen recognised by this antibody was shown to be Other topoisomerase II␣.20 monoclonal antibodies to this enzyme have since been developed, which yield clearer and more reproducible results than the original marker (figure 2).20 Figure 3. Immunohistochemical staining of Hodgkin’s cells and lymphocytes (nodular sclerosis). In With these antibodies, we showed in this cancer type, which can be successfully treated with regimens such as ABVD (doxorubicin, extensive retrospective studies, bleomycin, vinblastine, dacarbacine), CEP (cyclophosphamide, etoposide, cisplatin), EVAP including multivariate analyses, that (etoposide, vinblastine, doxorubicin, prednisone), or BEACOPP (bleomycin, etoposide, doxorubicin, topoisomerase II␣ is a reliable cyclophosphamide, vincristine, procarbazine, prednisone), topoisomerase II␣ and Ki67 are indicator of tumour aggressiveness. expressed in up to 90% of Hodgkin’s cells. Antibody KiS2, specific for S to M phase, stains only up to 30% of Hodgkin’s cells and few lymphocytes. Antibodies to topoisomerase I (C21; YC Cheng, Expression of the enzyme Yale University) stain all Hodgkin’s cells as well as the majority of surrounding lymphocytes. independently predicted the survival of patients with soft-tissue sarcoma42 specific but reflects malignancy in several ways. For and was the second strongest prognostic factor (after nodal instance, the expression of topoisomerases is commonly status) for both overall and metastasis-free survival in a upregulated in malignant transformed cells, probably cohort of 863 patients with infiltrating breast cancer.43 In a through deficiency in two negative regulators of series of 356 node-negative invasive mammary carcinomas, topoisomerase II expression and activity, p53 and pRB, topoisomerase II␣ and tumour size were the only which are known to be deleted or inactivated in many independent prognostic factors for overall and disease-free human cancers. Furthermore, loss of p53 function is likely survival.44 to impede the detection of DNA damage and to abrogate one important apoptotic pathway, thus allowing the DNA topoisomerase II as a target of survival of genetically degenerate cells. This mechanism chemotherapy may be another way in which the combination of increased Topoisomerase II is such a successful drug target because it topoisomerase II activity with defective p53 function may has an indispensable function in cell biology and it lacks foster genetic instability. Accordingly, accessory aberrations biological redundancy. Inhibitors of this enzyme have are found in association with deregulated topoisomerase become central parts of both primary and adjuvant expression. By analysis of a cohort of 230 patients with chemotherapy regimens in neoplastic diseases, and they breast cancer, Järvinen and colleagues showed that probably will remain so for the foreseeable future.45 topoisomerase II␣ is associated not only with proliferation, Although they form several distinct substance groups, they but also with absence of oestrogen receptors, progesterone are best categorised in two classes according to their receptors, or both, high malignancy grade, DNA biological properties—the topoisomerase poisons and the aneuploidy, and overexpression of HER2/neu catalytic inhibitors. Though as yet unproven, the common belief is that the ␣ isoform is the main pharmacological oncoprotein.37 As an alternative mechanism of topoisomerase IIα target in cancer cells. However, several studies point to an overexpression, amplifications of the coding gene have been additional response of topoisomerase II to cytotoxic reported. These occur, for instance, in association with therapy. amplifications of the HER2/neu gene, in non-small-cell lung carcinoma and breast cancer.38,39 Since the HER2/neu and The topoisomerase II poisons TOP2␣ genes are close to each other on the long arm of Topoisomerase II-poisons are defined as drugs that stabilise chromosome 17, amplification of one gene locus could affect the cleavable complex to cause DNA breaks (figure 1). They
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are called poisons because they convert this essential nuclear enzyme into a lethal poison.9 However, the pathway from DNA breaks to cell death by necrosis or apoptosis is still insufficiently explored.46 During the catalytic cycle of topoisomerase II, gaps in the DNA double strand in the form of cleavable complexes (CC) are present before (CC1; in figure 1 stage 3) and after (CC2; stage 4) DNA processing, exposing a weak point to the attack of intercalating substances. Of these substances, the anthracycline, daunorubicin, stabilises CC1, whereas amsacrine stabilises CC2, and etoposide acts on both CC1 and CC2. These different mechanisms of action nevertheless abut on a common endpoint since they all interfere with the re-ligase activity of topoisomerase II and thus perpetuate the DNA nicks that are thought to be the cause of apoptosis. Such strand breaks are thought to trigger the activity of caspases, which are in charge of programmed cell death.47 However, apoptosis could also be a consequence of aberrant non-homologous recombination after DNA damage48 or oxidative stress induced by topoisomerase poisons.49 Yet another poorly recognised mechanism is the downregulation and caspase-mediated proteolysis of protein kinase C␦ by etoposide targeting of the cytoplasmic fraction of topoisomerase II.50,51 Clearly, therefore, a definite danger lurks behind topoisomerase-directed therapy regimens. Indeed, many tumour cells are defective in apoptotic mechanisms, which may be related to inadequate detection of DNA damage (eg, by mutation of the ATM [ataxia-teleangiectasia-mutated] or p53 genes), deteriorated pathways signalling for cell-cycle arrest or cell death (involving pRB and p53 as higher-order regulators), or overexpression of antiapoptotic proteins such as BCL-2. Topoisomerase II itself may contribute to the disruption of at least one of these mechanisms, since it can break DNA in the region of chromosome 11q23 that includes the ATM gene.52 Such disturbances of normal control mechanisms are likely to allow survival of cells after illegitimate DNA recombination or inaccurate redistribution of daughter chromosomes under the influence of topoisomerase II poisons.36 In this way, not only the selection of more aggressive clones, but also the formation of secondary malignant disorders may be side-effects of a therapy involving topoisomerase poisons. The most frequent therapy-related neoplasias are acute leukaemias.53,54 Hence, neoplastic cells must be killed as efficiently as possible while the minimum injury is inflicted on untransformed cells. This aim can be approached in several ways: by identification of the tumours that would be expected to respond to topoisomerase II-poisons; by reduction of the toxic side-effects through combined regimens; and by more precise selection of the drug target. There is accumulating evidence that the sensitivity of tumour cells to topoisomerase poisons is proportional to their enzyme content.55–57 This idea may seem paradoxical because it implies a diminished drug-to-target ratio. The argument in favour of such a relation is, however, that higher expression of topoisomerase II will result in increased cleavage activity, and that the number of DNA strand breaks arising may be a critical determinant of cell death. Thus, for
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Review instance, coamplification of the HER2/neu and TOP2␣ genes is associated with increased sensitivity to amsacrine39 and anthracyclines,58 whereas deletion of topoisomerase II␣ in HER2-amplified breast tumours induces primary resistance to anthracyclines.58 The assessment of both HER2/neu and topoisomerase II␣ expression might therefore allow selection of patients who would benefit from combined treatment with trastuzumab (Herceptin) and a topoisomerase II␣-poison, whereas little benefit would be expected from the latter if the TOP2␣ gene were deleted. Transfection of heregulin 2 into breast-cancer cells produces a striking increase in their sensitivity to doxorubicin and etoposide.59 An analogous effect was observed when Ewing’s sarcoma cell lines were transfected with the E1A gene, which simultaneously resulted in downregulation of HER2/neu and upregulation of topoisomerase II␣.60 These observations suggest a close relation between the expression of the two genes. A problem in therapeutic designs nevertheless arises from the fact that the tumour aggressiveness associated with high proliferative activity generally over-rides the cell killing achieved by cytotoxic therapy.43 Surprisingly, the expression of topoisomerase II seems to be almost as important as that of the ␣ form for the sensitivity of malignant cells to topoisomerase II inhibitors. Studies on AML blasts from 72 patients revealed no correlation between topoisomerase II␣ activity and sensitivity to topoisomerase II-poisons, although activity of this isoenzyme in the tumour cells could be directly inhibited by incubation with the drugs in vitro. By contrast, topoisomerase II activity, which was not greatly inhibited by these compounds, was inversely correlated with the sensitivity of the cells. These findings were significant for idarubicin (p=0·017) and daunorubicin (p=0·006). Accordingly, resistant cells from patients (IC50 median) had the highest topoisomerase II activity. Cells from patients who relapsed after initial treatment with anthracyclincontaining regimens had a significantly decreased ratio of topoisomerase II␣/ activity than those who did not relapse (p=0·0276).61 The biological background of this intriguing finding nevertheless needs further investigation. Differential targeting of the two isoforms may play an important part in the action of topoisomerase II-poisons. Thus, etoposide,62 mitoxantrone, and amsacrine and its carbamate analogues16 act on both enzymes. Amsacrine seems to inhibit topoisomerase II preferentially,60 and there is evidence that low or absent expression of this isoform may result in very low sensitivity to amsacrine and related compounds.63 Other drugs have been developed that either target both isoenzymes, as chloroquinoxaline sulphonamide does,64 or specifically inhibit topoisomerase II, such as XK469.65 Because the specific functions of topoisomerase II are largely unknown, general application of drugs directed against this isoform may be difficult to envisage. However, the usefulness of such compounds is evident for tumours in which mutant forms of topoisomerase II␣ elude the effect of inhibitors.63 Moreover, the large fraction of G0/G1 cells found in most solid tumours66,67 may be sensitive to topoisomerase
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Review II inhibitors, as shown by the in vitro efficacy of XK469 as a solid-tumour-selective agent.68
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effectors of apoptosis. These properties may acquire clinical significance when classic treatment protocols are complicated by drug-resistance syndromes.
The topoisomerase II catalytic inhibitors In contrast to the poisons, the catalytic inhibitors do not induce DNA breaks and may even prevent their formation. They act at any stage in the enzymatic cycle other than the cleavable complex (figure 1), either by interference with topoisomerase II binding to DNA, or by locking the DNAbound enzyme in the shape of a closed clamp. Hence, two new therapeutic approaches based on the combination of a poison with a catalytic inhibitor in cancer treatment have been designed. Pharmacological modulation of a poison by a catalytic inhibitor
Since a catalytic inhibitor can abolish the toxic effect of a poison, this type of inhibitor can be used to direct the toxic effect. For example, the hydrophilic bisdioxopiperazine dexrazoxane (ICRF-187), which does not cross the bloodbrain barrier, can be used to increase the tolerated dose of etoposide, which does.69 This principle is currently being studied in a phase I/II trial in patients with metastases to the central nervous system from small-cell lung cancer. Another indication of counteracting a topoisomerase II poison with a catalytic inhibitor is given in the treatment of accidental anthracycline extravasation, in which the severe tissue necrosis can be significantly attenuated by use of dexrazoxane.70 A potentially much broader use of combining a protective catalytic inhibitor with a poison is suggested by the physiological propensity of most solid tumours to have acid extracellular environments. Weak bases cannot permeate the plasma membrane, but the neutral drug etoposide can; therefore, a non-toxic weakly alkaline catalytic inhibitor would be able to protect the normal tissues in the body without affecting the antitumour efficacy of etoposide. This idea has been successfully demonstrated in preclinical work with the weak base chloroquine, a malarial drug that is also a topoisomerase II catalytic inhibitor.71 However, chloroquine is too toxic for in vivo use, so new agents with the desired properties will have to be developed. Sequential use of a catalytic inhibitor with a poison
The lack of cross-resistance to catalytic inhibitors such as bisdioxopiperazines in cell lines selected for resistance to topoisomerase II-poisons makes the use of bisdioxopiperazines in the second-line treatment of malignant tumours a promising idea. In Japan, sobuzoxane (MST-16) has been approved for clinical use, which emphasises the pharmacological potential of the bisdioxopiperazine family in clinical settings. There are nevertheless exceptions to the suggestion of an alternative use of topoisomerase II catalytic inhibitors in tumour therapy. Thus, experimental compounds dexrazoxane and merbarone can induce apoptosis in the absence of DNA strand breaks.72 In both instances, this process seems to be related to the activation of caspase-like and ICE/CED3-like proteases, which are known to be distal
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Drug resistance The emperor of Pont, Mithridate VII (120–63 BC), intended to escape assassination by accustoming his metabolism to several popular poisons. By habitual intake of slowly increasing doses, he managed to become insensitive to these drugs. He was nevertheless successfully killed by administration of a substance he had neglected. This principle of developing and circumventing drug resistance is similar to that used today in experimental research and clinical therapy. Indeed, cells grown in the presence of increasing concentrations of cytotoxic drugs may become refractory not only to the substance applied, but also to structurally unrelated compounds, acquiring the multidrugresistance phenotype. Multidrug resistance to topoisomerase II poisons exists in two major forms: one is attributable to efflux pumps in the cell membrane that lower the steady-state concentration of the drug at its target site (transport or target multidrug resistance); in the other form, the activity and sensitivity of the target enzyme topoisomerase II itself are decreased by downregulation or mutation (altered topoisomerase II multidrug resistance). The classic multidrug-resistance phenotype, whereby a cell becomes resistant not only to the selecting drug but also to structurally unrelated drugs, has stimulated a tremendous amount of research activity, which has been reviewed elsewhere and is beyond the scope of this review.73,74 The altered topoisomerase II multidrug-resistance phenotype, associated with a reduction in topoisomerase II content and activity, was first described in 1986 and 1988.75,76 That this phenotype could also be caused by a mutation in topoisomerase II was discovered by Hinds and colleagues in 1991.77 Subsequently, other mutations in human topoisomerase II were shown to be functional in a transfected yeast system.78 The mutations inducing resistance to topoisomerase II poisons cluster around the Walker B consensus ATP-binding site or around the Y805 catalytic tyrosine,79 whereas mutations causing resistance to the bisdioxopiperazine type catalytic inhibitors cluster in and around the Walker A ATP-binding site.80 These observations further attest to the fundamental difference between these two classes of compounds. From a clinical perspective, the significance of the multidrug-resistance phenotype is difficult to assess. Studies on the expression of drug pumps, which are mostly done by immunohistochemistry, in many, but by no means all, cases detect a correlation between overexpression of a pump and clinical resistance. P-glycoprotein expression has been studied to such an extent that two meta-analyses have been done, one on AML81 and one on breast cancer,82 both documenting for this pumps relevance in clinical drug resistance. The altered topoisomerase II multidrugresistance phenotype has been much less studied because changes in topoisomerase II expression in clinical material have long been difficult to measure. However, mutations in
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Search strategy and selection criteria Published data for this review were searched by using Current Contents and PubMed; historical data (before the 1960s) were found by secondary citation. Articles were chosen by their relevance and only those published in English were selected. We also contacted researchers to obtain unpublished data.
topoisomerase II do not seem to have a major role in resistance; the few studies so far found no mutations83,84 or a low frequency (eg, one in 13 cases85). The H69/VP etoposide-resistant cell line, which overexpresses both P-glycoprotein and multidrugresistance-associated protein, another transmembrane pump, was eventually shown to have the altered topoisomerase II multidrug-resistance phenotype, with a splice-site mutation in topoisomerase II.86 Moreover, this H69/VP strain shows moderate overexpression of lung resistance protein, but not of breast-cancer resistance protein or mitoxantrone-resistance protein (unpublished). That a single drug-selected resistant cell line can simultaneously display four of the five known resistance phenotypes highlights the complexity that bedevils the clinical application of the information on multidrug resistance collected through decades of laboratory research. However, this experience may be helpful in selection of new agents destined to circumvent the various multidrugresistance phenotypes by being poor substrates for the efflux pumps, or like topoisomerase II catalytic inhibitors, by attacking the target in a different way.
Conclusions Topoisomerase II-targeting drugs have been used extensively in cancer chemotherapy since the clinical introduction of daunorubicin in 1964, and will continue to be used for the foreseeable future. Although the new cytostatic drug classes, such as those that inhibit signal transduction, telomerase, angiogenesis, or proteases, will be of increasing real benefit to cancer patients, current preclinical and clinical studies show that cytostatic agents generally have to be combined with cytotoxic drugs to achieve their full efficacy. The vast amount of knowledge and experience gained over the past four decades in the use of topoisomerase II anticancer agents should aid in developing even better ways of attacking this specific drug target. Acknowledgments
We thank Bernd Wüsthoff and Angela Kellner for helpful discussions during the preparation of this paper. References
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Review
DNA topoisomerase II in oncology
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