Summary Topoisomerase enzymes - found in prokaryotes to human cells - control conformational changes in DNA and aid the orderly progression of DNA replication, gene transcription and the separation of daughter chromosomes at cell division. Several classes of anti-cancer drugs are now recognised as topoisomerase poisons because of their ability to trap topoisomerase molecules on DNA as ‘cleavable complexes’. Understanding how drugs generate such complexes and why they are toxic to actively growing cancer cells is a major challenge for the development of modern approaches to chemotherapy. Introduction Ideally, anti-cancer chemotherapy should aim to spare normal tissue while eradicating tumor cells and circumventing their predilection to develop drug resistance. Recent studies have established that a critical action of several apparently unrelated anticancer drugs is to disturb or ‘poison’ the molecular embrace of the double helix by certain ty es of nuclear enzymes - the DNA topoisomerases(’-#. These discoveries have provided valuable insights into how some anti-cancer drugs actually work and also why they may fail despite efficient delivery to neoplastic cells. Topoisomerase poisons also present us with a means of dissecting the mode of action and the cellular roles of this intriguing class of nuclear enzymes. This mini-review describes the eukaryotic DNA type I and type I1 topoisomerases (the human enzymes referred to as topoisomerase I and I1 respectively), highlighting recent ideas about topoisomerase functions and the mechanisms of enzyme poisoning. Modus Operandi The double helical structure of DNA can present problems to the unwary mammalian cell attempting to pack, replicate or transcribe DNA, because of the ability of DNA to supercoil, knot and interlink (Fig. 1). Furthermore, the topological state of DNA may itself be important to a specific process such as recombination or in determining the probability with which a regulatory protein binds at a given site on DNA. The DNA in

eukaryotic cells acts topologically in a fashion similar to that of covalently closed duplex circles since the DNA is effectively constrained by anchorage proteins - forming a nuclear matrix - organising DNA into chromatin loop domains. The topological state of an anchored DNA loop (or circle) that is neither knotted nor interlinked can be described by the linking number, essentially the number of times the two strands of the helix are wound around each other. Underwinding results in negative supercoiling (as observed in partially de-proteinised DNA isolated from mammalian cells) whereas overwinding results in positive supercoiling. Changing the conformation of chromatin domains by co-ordinating a process of strand breaking, passing and ligation is the responsibility of the nuclear located topoisomerase enzymed4). Type I enzymes operate by breaking a single strand of DNA, passing the intact strand through the resulting gap and resealing the gap in the DNA backbone, thereby changing the overall extent of strand interwinding. If this process of strand passing is carried out repetitively then even highly supercoiled DNA can be relaxed, the linking number changing at each step by a decrement of +1. Type I1 enzymes can also effect supercoil relaxation by passing an intact duplex through a double strand gap, the linking number of the DNA changing by multiples of two. The figure shows how the topoisomerases can deal with various conformational changes to DNA by forming a complex with the helix, which is in equilibrium with a covalently bound ‘cleavable complex’ sequestering strand breaks. Supercoiled DNA can be relaxed either by the energy-independent single-strand passing activity of topoisomerase I or by the double-strand passing activity of topoisomerase 11. Topoisomerase I1 can additionally effect unknotting and decatenation (unlinking) of interlinked circles of DNA. The activities of the type I1 enzyme require ATP hydrolysis for the protein to be released from DNA and to be available for another strand-passing cycle. Forms Human topoisomerase I is a 100 kd monomeric protein derived from a single copy gene. Results so far indicate that the gene contains several intervening sequences and is located on human chromosome 2O(’). Eukaryotic DNA topoisomerase I can show tight noncovalent association at a high affinity binding sequence on DNA, with the enzyme protecting both strands over a 2G-base pair re ion in which the cleavage site is centrally locatedb). However, for the eukaryotic DNA topoisomerase I cleavage reaction, topology may be more critical than sequence effects, in line with a regulatory Unlike function of the enzyme in gene tran~cription(~). the type I enzyme, topoisomerase I1 appears to have been highly conserved throughout evolution and is the eukaryotic counterpart of the bacterial DNA gyrase enzyme. The human topoisomerase I1 single cop gene has been cloned and mapped to chromosome 17(t7). The

Generation of topological Resolved products

Supercoiling

Knotting

Interlinking

Resolved products

A : relaxation B : unknotting C : decatenation

A : relaxation

non-cleavable complex

cleavable complex

CPT

7

7

A

JVP-16

Presence of opoisomerase poison

I

I

I

I

I

1

Alkali

Interaction with

I

$-

1

and transcription

single + double strand breaks

single strand breaks

amino acid sequence of the human 170 kd homodimeric enzyme indicates the presence of a 'leucine zipper', a novel motif found in several nuclear located proteins(') and probably im ortant to the normal functions of the type 11 enzyme(' ).

t:

-

Topoisomerase Poisoning Trapping the Cleavable Complex Several classes of topoisomerase inhibitors have been identified (Table 1). However, only certain antitumor

Fig. 1. Poisoning of the topoisomerase cycles of mammalian cells by antitumor drugs. See text for discussion of details.

agents, such as the epipodophyllotoxins (eg VP-16), are capable of trapping the cleavable complex of topoisomerase I1 in a nonproductive or stabilised form (Fig. 1). This stabilised complex has unusual properties; upon denaturing the trapped protein with agents such as sodium dodecyl sulphate (SDS) or alkali, DNA strand breaks are revealed with protein covalently linked to the 5' phosphoryl end of each broken strand("). Both single- and double-strand breaks can be revealed, with the latter structure showing a recessing of the 3' hydroxyl end by four bases. Initially it was thought that

Table 1. Inhibitors of D N A topoisomerases" Class of drug Type I1 enzyme inhibitors: Coumarins Quinolones Acridines Anthracyclines Anthraquinones Ellipticines Epipodophyllotoxins Type I enzyme inhibitors: Plant alkaloids Type I and type I1 enzyme inhibitors: DNA minor groove binders

Example

Target enzyme

Novobiocin Nalidixic acid Amsacrine Adriamycin Mitoxantrone 2-Me-9-OH-E+ W-16

Bacterial gyrase (A subunit) Bacterial gyrase (B subunit) Eukaryotic topoisomerase 11* Eukaryotic topoisomerase 11* Eukaryotic topoisomerase 11* Eukaryotic topoisomerase 11* Eukaryotic topoisomerase 11*

Camptothecin

Eukaryotic topoisomerase I*

Distam ycin Hoechst dyes

Eukaryotic topoisomerase I and I1

aTable modified from ref. 3. *Enzyme trapped as a cleavable complex.

the epipodophyllotoxins did not bind to DNA, providing a case for the existence of drug binding sites on the topoisomerase I1 molecule(2)as suggested diagrammatically in the figure. However, using experimental conditions designed to detect limited numbers of DNA binding sites, it has been shown(12)that epipodophyllotoxins can indeed bind to DNA, with greater drug binding to single-stranded DNA than to doublestranded linear or supercoiled DNA. Many antitumor DNA intercalating drugs (e.g. the aminoacridine amsacrine; Table 1) can also trap cleavable complexes in a manner which is not merely a reflection of the disruption of DNA structure by the process of intercalation per se(1,13).It is possible that the topoisomeraseDNA complex itself generates a novel binding site for the drug molecule on DNA, providing a general mechanism whereby intercalating agents and epipodophyllotoxins alter topoisomerase function and presumably exert their shared antitumor effects. Topoisomerase I can also be trapped as a cleavable complex (Fig. 1) by a specific DNA topoisomerase I poison, the plant alkaloid camptothecin (CPT). Treatment of the stabilised complex with a strong protein denaturant results in the cleavage of a single phosphodiester bond and covalent linking of the protein to the 3'phosphoryl end of the broken strand via a tyrosyl phosphate bond('). Studies on several new camptothecin derivatives indicate that antitumor activity is related to inhibition of topoisomerase I(14). Locations and Roles Roles can be implied from the strategic disposition of enzyme molecules, topoisomerase I being abundant in actively transcribing regions and topoisomerase I1 being distributed more uniformly along chromosomes but concentrated at the base of chromatin loop domains. Antibody probes have been used to examine the expression of DNA topoisomerases I and I1 throughout the cell cycle(15).Topoisomerase I shows no significant fluctuations in content or stability across the cell cycle.

In contrast, topoisomerase I1 undergoes significant cell cycle-dependent alterations in both amount and stability. As cells progress from mitosis into G I , much of the topoisomerase I1 is degraded. During the first 2 h of G I , the half life of topoisomerase I1 is decreased from that measured in asynchronous cell populations by a factor of seven. Topoisomerase poisons are becoming invaluable probes for highlighting the involvement of these enzymes in specific cellular processes. For example, camptothecin inhibits both DNA and RNA synthesis. Although the implied role of topoisomerase I function in DNA replication is still not clear(') there is a growing body of evidence linking the type I enzyme activity with efficient transcription. Indeed, topoisomerase I and RNA polymerase I are found tightly complexed both in vivo and in vitro(16)and transcription is emerging as one of the principal factors affecting intracellular DNA supercoiling. Consider the problem of translating an RNA polymerase molecule along the DNA helix there could be a bow wave of positive supercoiling generated ahead of the transcription com lex and a wake of negative supercoiling to the rear(' ). Support for this twin supercoiled domain model comes from the use of prokaryotic topoisomerase I as a tool for the selective removal of negative supercoils in DNA that is being transcribed from a single promoter in vitrd"); under such conditions, transcribing DNA shows extensive positive supercoiling. An active role for the type I enzyme in supercoil relaxation during transcription is also consistent with the rapid, reversible arrest of ribosomal RNA synthesis by camptothecin and its correlation with the presence of trapped complexes within the transcribed region of human ribosomal RNA genes. The distribution of RNA polymerase molecules along the transcription unit of human ribosomal RNA genes in camptothecin-treated human cells suggest that DNA topoisomerase I is normally involved in the elongation step of transcription(18). The type I1 enzyme is essential for the segregation of replicated daughter chromosomes(19). Recent exper-

P

iments with yeast cells(20)support the hypothesis that the mitotic spindle is necessary to allow topoisomerase I1 to complete the untangling of sister chromatids. The chromosome condensation/decondensation cycle itself appears to be coupled to a parallel cycle of synthesis and degradation of this enzyme(21). Not surprisingly there is evidence, at least in lower eukaryotes, that the type I1 enzyme can compensate for deficiencies in the functions of the type I enzyme(4). However, it is not clear what penalties a human cell must bear for such putative molecular substitution when asked to cope with adverse environments such as those arranged by the chemotherapist administering topoisomerase poisons. A role of topoisomerases in the regulation of DNA repair has long been an attractive concept. However it has been surprisingly difficult to obtain direct evidence of such a role in mammalian cells and this subject has been dealt with in a previous BioEssays mini-review(22). It is possible that topoisomerases may be recruited into DNA repair pathways as ‘innocent bystanders’ as the result of some general response to DNA damage. For example, the presence of strand breaks in cellular DNA is known to stimulate a distinct modification, poly(ADP-ribosylation), of chromosomal proteins. These modifications affect the structural and functional properties of chromatin includin the coordination of the repair of the inducing lesionsfi3). The poly(ADPR) substitution of topoisomerase I is elevated by a factor of 6-10 from the steady-state level of 0.1 % when mammalian cells are exposed to an agent generating active DNA-damaging radicals(24). Thus the inactivation of topoisomerase I by modification in the neighbourhood of DNA breakage may temporarily shut down DNA replication and allow DNA repair to occur. Why Do Topoisomerase Poisons Poison? There are two views of the cellular consequences of topoisomerase poisoning. Firstly, that the trapping of the enzyme in a redundant state for a period of time results in the withdrawal from service of a critical cellular enzyme. The enzyme may be deemed to be critical because the cell has not been given the opportunity to compensate for the unexpected loss of function. Secondly, and not necessarily independently, the cleavable complex itself may be a lethal lesion. The second view is currently favoured but suggests that either the complex generates some action which is deleterious to the cell or that the complex interacts with some other cellular factor which leads to cell death. As far as the effects of topoisomerase poisoning are concerned, the ‘when and where’ appear to be just as important as the ‘why and how’.’ In the case of camptothecin, toxicity can be blocked by the co-administration of a DNA synthesis inhibitor suggesting that the collision of a replication fork with a trapped topoisomerase I complex is a lethal event“). The current models for topoisomerase 11-related

lethality are less straightforward. The toxicity of a type I1 enzyme complex may relate to its position within the genome, for example clustering of topoisomerase I1 cleavable complexes can occur in sequences adjacent to an active gene(25).Additionally, a family of A+T-rich sequences termed MARs (‘matrix association regions’), known to mediate chromosomal loop attachment, both specifically bind and contain multiple sites of cleavage by topoisomerase 11. There is the suggestion that dysfunctioning MARs may be sites of topoisomerasedriven illegitimate recombination associated with the breakpoint of a chromosomal translocation(26). Newly replicated DNA is a preferential target for antitopoisomerase I1 drugs(”) but the question arises whether factors in addition to cleavable complex formation in a ‘sensitive’ region are needed to express lethality. Recently it has been shown that in short term experiments the inhibition of RNA or protein synthesis protects cells from the cytotoxic action of the topoisomerase I1 poison amsacrine without reducing DNA breakage, cleavable complex formation or DNA topoisomerase I1 activity(28).The search for additional labile protein factors which convert cleavable complexes into lethal lesions has begun, with the hope that such factors can be manipulated to the chemotherapist’s advantage. The majority of complexes induced by agents such as VP-16 and amsacrine disappear rapidly from cellular DNA(2)and the key to the action of topoisomerase I1 poisons is the nature of the conversion of complexes into lethal lesions. An attractive candidate for the form of the converted complex is a persistent DNA double strand break. Clues have been provided by a human VP-16- and amsacrine-sensitive cell line which overexpresses DNA topoisomerase 11(29)and shows elevated levels of drug-induced complexes. The over-producer also shows increased levels of other lesions (single and double strand breaks) detectable b a method which is not responsive to intact complexes(-”). This is consistent with an increase in complex formation increasing the background level of lesions which have undergone conversion. A similar observation(31) has been made following treatment of topoisomerase I1 over-producing cells with a Hoechst dye, a DNA minor groove binding drug which can modify topoisomerase function without enerating a classical cleavable complex (Table This suggests that disturbance of topoisomerase function is the essential factor rather than the formation of the cleavable complex per se. Perhaps a new form of potentially lethal lesion should be proposed - the convertible complex. The mechanism of conversion can only be speculated working with bacteriophage upon. Ripley et DNA in amsacrine-induced frame-shift mutagenesis experiments have proposed models which involve DNA polymerase and/or exonuclease reactions resulting in the deletion or duplication of a small number of bases adjacent to the cleavage site of a trapped complex. Accordingly, complex-conversion in human cells could involve an array of DNA metabolising enzymes and

7

serve as a vehicle for the generation of chromosomal aberrations, sister chromatid exchanges and genetic recombination Manipulating Topoisomerase Activity in Tumor Cells Topoisomerase I1 appears to be a good candidate for therapeutic manipulation. Non-proliferating tumor cells will be naturally resistant to topoisomerase poisons whereas coaxing cells to increase their levels of topoisomerase I1 will enhance their sensitivity(29).In serumstarved cultured human cells, the levels of the enzyme can be increased from lo4 copies/cell to lo6 copies/cell by the addition of serum to the growth medium. In contrast, the intracellular levels of DNA topoisomerase I are largely unaffected by growth conditions(36).The link between growth factor stimulation of a cell population and the enhanced availability of DNA topoisomerase I1 target molecules offers the daunting possibility of optimising chemotherapy by manoeuvres aimed initially at stimulating the expression of this cell cycleregulated enzyme in targeted cells! For example, estrogen stimulation of human breast cancer cells enhances the induction of DNA cleavage by the topoisomerase I1 poisons VP-16 or amsacrine with a corresponding enhancement of cell killing(37).Increased cellular content of the target enzyme could be achieved at least 4 h prior to the enhancement of DNA synthesis or active cell cycle traverse(38)generating a window of sensitivity in non-proliferating cells. Estrogen stimulation of receptor-positive breast cancer cells may prove to be a clinically relevant strategy for improving the selectivity and cytotoxicity of some topoisomerase 11 poisons(37). Novel Pathways for Drug Resistance in Cancer Cells It has been noted that nuclear and cytoplasmic type I topoisomerase-specific activities are higher in transformed cell lines than in the normal counterparts(”). Furthermore, control of topoisomerase I1 stability is altered upon transformation(40). Such putative differences between normal and tumor tissue could affect drug sensitivity. However, topoisomerases can be involved in drug resistance in several ways. They are nuclear-located targets and can be afforded an unusual degree of target protection by the effects of the rapid cellular efflux of xenobiotic compounds achieved by multidrug resistant cells over-expressing a specific membrane p-gly~oprotein(~~). Alternatively, a cell may reduce the number of enzyme molecules or modify the intrinsic sensitivity of the protein to a topoisomerase poison(2.42.43) Changes in the cleavable complex conversion pathway may also give rise to resistance to poisons. Proteins which alter topoisomerase function may vary in tumor cells and provide a second generation of tar ets for chemotherapy. An example is the discoveryb4) that

murine mastocytoma cells contain a labile protein factor that enhances formation of amsacrine-induced topoisomerase 11-DNA complexes and that continuous protein synthesis is required to maintain the factor. This appears to be the first direct evidence of a protein factor that modulates drug-induced topoisomerase I1 poisoning. Perhaps topoisomerases will be shown to have unexpected roles in resistance to DNA damaging agents other than topoisomerase poisons once we have identified the full array of repair pathways available to mammalian cells. A new topoisomerase-dependent repair pathway has recently been identified in a mutant mouse cell line for the rapid ejection of non-covalently bound drug molecules from the minor groove of DNA(45).The topoisomerase I1 inhibitors novobiocin, VP-16, nalidixic acid and the topoisomerase I-poison camptothecin can inhibit the rapid ejection process(%). This topoisomerase dependency is consistent with the ability of minor groove binders to distort the manner in which DNA associates with nucleosomal core part i c l e ~ ( ~since ~ ) , such imposed torsional stress could be subject to the action of cellular topoisomerases. The minor groove binders are potential DNA sequence targeting moieties for future generations of antitumor agents and here we have glimpsed how topoisomerases may facilitate the expression of novel modes of drug resistance. Future Perspectives Not knowing the full nature of the cytotoxic interaction between an antitumor agent and its target cell has always undermined our ability to design more effective drugs, to target treatment modalities, and to gain some therapeutic advantage by manipulating the biology of neoplastic cells in situ. This situation has now changed for several classes of potent antitumor agents which poison DNA topoisomerases. Tumor cells carry the seeds of their own destruction in the form of these nuclear enzymes - it is up to the chemotherapist to take advantage of this unique opportunity. References 1 LIU,L. F. (1989). DNA topoisomerase poisons as antitumour drugs. Annu. Rev. Biochem. 58, 351-375. 2 GLISSON, B. S . AND Ross, W. E. (1987).DNA topoisomerase 11: A primer on the enzyme and its unusual role as a multidrug target in cancer chemotherapy. Pharmac. Ther. 32, 89-106. 3 DRLICA, K. AND FRANCO, R. J. (1988). Inhibitors of DNA topoisomerases Biochemistry 27, 2253-2259. 4 WANC. .I.C. (1985). DNA topoisomerases. Annu. Rev. Biochem. 54, 665-697. 5 ZHOU.B. S . , BASTOW, K. F. AND CHENC,Y. C. (1989). Characterization of the 3’ region of the human DNA topoisomerase I gene. Cancer Res. 49, 3922-3927. 6 STEVNSNER, T., MORTENSEN, U. H., WESTERGAARD, 0. A N D BONVEN, B. J. (1989). Interactions between eukaryotic DNA topoisomerase I and a specific binding sequence. J . B i d . Chem. 264, 10110-10113. 7 CAMILLONI, G.,DIMARTINO, E., DIMAURO,E. A N D CASERTA, M . (1989). Regulation of the function of eukaryotic DNA topoisomerase I: topological conditions for inactivity. Proc. Nad. Acad. Sci. USA 86, 3080-3084. 8 TSAI-PFl.UGFEI.DER, M., LIU, L. F., LIU, A. A,. TEWEY,K. M . , WHANG-

PENG,J., KNUTSEN, T., HUEBNER, K., CROCE,C. M. A N D WANG,J. C. (1988). Cloning and sequencing of cDNA encoding human DNA topoisomerase I1 and localization of the gene to chromosome region 17q21-22. Proc. Nurl. Acud. Sci. USA 85, 7177-7181. 9 LANDSCHULTZ, W. H., JOHNSON, R. F. AND MCKNIGHT, S . L. (1988). The leucine zipper: A hypothetical structure common to a new class of DNA binding protein. Science 240, 1759-1765. 10 ZwELLiNc, L. A. AND PERRY, W. M. (1989). Leucine zipper in human DNA topoisomerase 11. Mol. Endocrinol. 3, 603-604. 11 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-anisidide and adriamycin. Biochemi.w-y 20, 6553-6563. 12 CHOW.K. C., MACDONALD. T. L. AND Ross, W. E. (1988). DNA binding by epipodophyllotoxins and N-acyl anthracyclines: implications for mechanism of topoisomerase I1 inhibition. Mol. Phurmucol. 34, 467-473. 13 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 cleavage activity from cell nuclei and identification as topoisomerase 11. Biochemistry 25, 9-16. 14 HERTZBERG, R. P., CARANFA,M. J., HOLDEN, K. G., JAKAS,D. R., 5. O., JOHNSON, R. GALLAGHER. G., MAITERN, M. R., MONG,S . M., BARTUS, K. AND KINGSBURY, W. D. (1989). Modification of the hydroxy lactone ring of camptothecin: inhibition of mammatian topoisomerase 1and biological activity. J . Med. Chem. 32,715-720. 15 HECK, M. M., HIITELMAN. w. N. A N D EARNSHAW, W. c. (1988). Differential expression of DNA topoisomerases I and I1 during the eukaryotic cell cycle. Proc. Nurl. Acad. Sci. USA 85, 1086-1090. 16 ROSE, K. M., SZOPA, J., HAN, F. S . , CHENG,Y. C., RICHTER, A. AND SCHEER, U . (1988). Association of DNA topoisomerase I and RNA polymerase I: a possible role for topoisomerase I in ribosomal gene transcription. Chromosoma %, 411-416. 17 TSAO, Y. P., Wu, H. Y. AND Liu, L. F. (1989). Transcription-driven supercoiling of DNA: direct biochemical evidence from in virro studies. Cell 56, 111-118. 18 ZHANG, H., WANG.J. C. A N D Liu, L. F. (1988). Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes. Proc. Nurl. Acud. Sri. USA 85, 1060-1064. 19 YANG,L., WOLD,M. S , Lr. J. J.. KELLY, T. J. AND Liu, L. F. (1987). Roles of DNA topoisomerases in simian virus 40 DNA replication in vibo. Proc. Nurl. Acad. Sci. USA 84, 950-954. 20 HOLM,C., STEARNS, T. AND BOTSTEIN, D. (1989). DNA topoisomerase I1 must act at mitosis to prevent nondisjunction and chromosome breakage. Mol. Cell. Biol. 9, 159-168. 21 HECK, M. M., HIITELMAN,W. N. AND EARNSHAW, W. C. (1988). Differential expression of DNA topoisomerases I and I1 during the eukaryotic cell cycle. Proc. Nurl. Acud. Sci. USA 85, 1086-1090. 22 DOWNES, C. S. A N D JOHNSON, R. T. (1988). DNA topoisomerases and DNA repair. BioEssays 8, 179-184. 23 SHALL,S. (1988). ADP-ribosylation of proteins: a ubiquitous cellular control mechanism. Adv. Exp. Med. Biol. 231, 597-611. 24 KRUPITZA, G. A N D CERUITI. P. (1989). ADP-ribosylation of ADPRtransferase and topoisomerase I in intact mouse epidermal cells JB6. Biochemisrry 28, 2034-2040. 25 Riou, J. F., MULTON,E., VILAREM, M. J.. LARSEN,C. J. AND RIOU,G. (1986). In viuo stimulation by antitumor drugs of the topoisomerase I1 induced cleavage sites in c-myc protooncogene. Biochem. Biophys. Res. Commun. 137, 154-160. 26 SPERRY, A. O., BLASQUEZ, V. C. AND GARRARD, W. T. (1989). Dysfunction of chromosomal loop attachment sites: illegitimate recombination linked to matrix association regions and topoisomerase 11. Proc. Nurl. Acud. Sci. USA 86, 5497-5501. 27 WOYNAROWSKI, J . M., SIGMUND, R. D. AND BEERMAN, T. A . (1988). Topoisomerase-11-mediated lesions in nascent DNA: comparison of the effects of epipodophyllotoxin derivatives, VM-26 and VP-16, and 9-anilinoacridine derivatives, m-AMSA and o-AMSA. Biochim. Biophys. Acru 950, 21-29. 28 SCHNEIDER, E., LAWSON, P. A. A N D RALPH,R. K. (1989). Inhibition of protein synthesis reduces the cytotoxicity of 4’-(9-acridinylamino)methanesulfon-m-anisidide without affecting DNA breakage and DNA

topoisomerase I1 in a murine mastocytoma cell line. Biochem. Pharmacol. 38, 263-269. 29 SMITH,P. J. AND MAKINSON, T. A. (1989). Cellular consequences of overproduction of DNA topoisomerase I1 in an ataxia-telangiectasia cell line. Cunrer Res. 49, 1118-1124. 30 SMITH,P. J . , ANDERSON, C. 0. AND WATSON, J. V. (1986). Predominant role for DNA damage in etoposide-induced cytotoxicity and cell cycle perturbation in human SV40-transformed fibroblasts. Cancer Res. 46. 5641-5645. 31 SMITH,P. J . (1984). Relationship between a chromatin anomaly in ataxia telangiectasia cells and enhanced sensitivity to DNA damage. Curcinogenesi,s5. 1345- 1350. 32 WOYNAROWSKI, J. M., MCHUGH, M., SIGMUND, R. D. A N D BEERMAN. T. A. (1989). Modulation of topoisomerase I1 catalytic activity by DNA minor groove binding agents distamycin, Hoechst 33258, and 4‘,6-diamidine-2-phenylindole. Mol. Phurmucol. 35, 177-182 33 RIPLEY, L. S., DUBINS, J. S . . DEBOER,J. G., DEMARINI, D. M.. BOCERD. A. K. N. (1988). Hotspot sites for acridine-induced frameshift M. AND KREUZER, mutations in bacteriophage T4 correspond to sites of action of the T4 type I1 topoisomerase. J . Molec. B i d . 200, 665-680. 34 LONG,B. H. AND STRINGFELLOW, D. A. (1988). Inhibitors of topoisomerase 11: structure, activity relationships and mechanism of action of podophyllin congeners. Adv. Enzyme Regul. 21,223-256. 35 ANDERSON,H. C. AND KIHLMAN,B. A . (1989). The production of chromosomal alterations in human lymphocytes by drugs known to intcrfcre with the activity of DNA topoisomerase 11. 1. m-AMSA. Curcinogenesi.c 10, 123- 130. 36 HSIANG, Y. H., WU, H. Y. AND Llu, L. F. (1988). Proliferation-dependent regulation of DNA topoisomerase I1 in cultured human cells. Cancer Res. 48, 3230-3235. 37 EPSTEIN, R. J . AND SMITH,P. J. (1988). Estrogen-induced potentiation of DNA damage and cytotoxicity in human breast cancer cells treated with topoisomerase 11-interactive antitumour drugs. Cancer Res. 48, 297-303. 38 EPSTEIN, R. E. AND SMITH, P. J. (1989). Mitogen-induced topoisomerase I1 synthesis precedes DNA synthesis in human breast cancer cells. Biochem. Biophys. Res. Commun. 160, 12-17. 39 CRESPI,M. D., MLADOVAN, A. G. AND BALDI,A . (1988). Incremcnt of DNA topoisomerases in chemically and virally transformed cells. Exp. Cell Res. 175, 206-215. 40 HECK, M. M., HIITELMAN,W. N. AND EARNSHAW, W. C. (1988). Differential expression of DNA topoisomerases I and I1 during the eukaryotic cell cycle. Proc. Nurl. Acud. Sci. USA 85, 1086-1090. 41 ENDICOIT,J. A. AND LING, v. (1989). ‘The biochemistry of P-glycoproteinmediated multidrug resistance. Annu. Rev Biochem. 58, 137. 42 GUPTA,R. S., GUPTA,R., ENG,B., LOCK,R. B.. Ross, W. E., HERTZBERG. R. P., CARANFA, M. J. AND JOHNSON, R. K. (1988). Camptothecin-reyistant mutants of Chinese hamster ovary cells containing a resistant form of topoisomerase I. Cancer Res. 48, 6404-6410. 43 KJELDSEN, E., BONVEN. B. J., ANDOH,T., ISHII.K.. OKADA, K., BOLUND, L. AND WESTERCAARD, 0. (1988). Characterization of a camptothecin-resistant human DNA topoisomerase I. J . Biol. Chem. 263, 3912-3916. 44 DARKIN, S . J. AND RALPH,R. K. (1989). A protein factor that enhances amsacrine-mediated formation of topoisomerase 11-DNA complexes in murine mastocytoma cell nuclei. Biochim. Biophys. Acru 1007, 295-300. 45 SMITH, P. J., LACY,M., DEBENHAM, P. G. AND WATSON, J. v. (1988). A mammalian cell mutant with enhanced capacity to dissociate a bisbenzimidazole dye-DNA complex. Carcinogenesis 9, 485-490. 46 SMITH,P. J., DEBENHAM, P. G. AND WATSON, J. V. (1989). A role of DNA topoisomerases in the active dissociation of DNA minor groove-ligand complexes: A flow cytometric study of inhibitor effects. Murur. Res. 217. 163- 172. 47 PORTUGAL L. J. AND WARING, M. J. (1986). Antibiotics which can alter the rotational orientation of nucleosome core DNA. Nucl. Acids Res. 14, 8735-8754.

Paul J. Smith is at the MRC Clinical Oncology and Radiotherapeutics Unit, MRC Centre, Hills Road, Cambridge, CB2 2QH, UK.