Stabilization of the topoisomerase II-DNA cleavage complex by antineoplastic drugs: inhibition of enzyme-mediated DNA religation by 4'-(9-acridinylamino)methanesulfon-m-anisidide

In order to elucidate the mechanism by which the intercalative antineoplastic drug 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) stabilizes the covalent topoisomerase II-DNA cleavage complex, the effect of the drug on the DNA cleavage/religation reaction of the type II enzyme from Drosophila melanogaster was examined. At a concentration of 60 microM, m-AMSA enhanced topoisomerase II mediated double-stranded DNA breakage approximately 5-fold. Drug-induced stabilization of the enzyme-DNA cleavage complex was readily reversed by the addition of EDTA or salt. When a DNA religation assay was utilized, m-AMSA was found to inhibit the topoisomerase II mediated rejoining of cleaved DNA approximately 3.5-fold. This result is similar to that previously reported for the effects of etoposide on the activity of the Drosophila enzyme [Osheroff, N. (1989) Biochemistry 28, 6157-6160]. Thus, it appears that structurally disparate classes of topoisomerase II targeted antineoplastic drugs stabilize the enzyme's DNA cleavage complex primarily by interfering with the ability of topoisomerase II to religate DNA.     

Effect of antineoplastic agents on the DNA cleavage/religation reaction of eukaryotic topoisomerase II: inhibition of DNA religation by etoposide

Beyond its essential physiological functions, topoisomerase II is the primary cellular target for a number of clinically relevant antineoplastic drugs. Although the chemotherapeutic efficacies of these drugs correlate with their abilities to stabilize the covalent topoisomerase II-DNA cleavage complex, their molecular mechanism of action has yet to be described. In order to characterize the drug-induced stabilization of this enzyme-DNA complex, the effect of etoposide on the DNA cleavage/religation reaction of Drosophila melanogaster topoisomerase II was studied. Under the conditions employed, etoposide increased levels of enzyme-mediated double-stranded DNA cleavage 5-6-fold and single-stranded cleavage approximately 4-fold. Maximal stimulation was observed at 80-100 microM etoposide with 50% of the maximal effect at approximately 15 microM drug. By employing a topoisomerase II mediated DNA religation assay [Osheroff, N. & Zechiedrich, E.L. (1987) Biochemistry 26, 4303-4309], etoposide was found to stabilize the enzyme-DNA cleavage complex (at least in part) by inhibiting the enzyme's ability to religate cleaved DNA. Moreover, in order for the drug to affect religation, it has to be present at the time of DNA cleavage.     

Effect of casein kinase II-mediated phosphorylation on the catalytic cycle of topoisomerase II. Regulation of enzyme activity by enhancement of ATP hydrolysis.

The catalytic activity of topoisomerase II is stimulated approximately 2-3-fold following phosphorylation by casein kinase II (Ackerman, P., Glover, C. V. C., and Osheroff, N. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3164-3168). In order to delineate the mechanism by which the activity of the enzyme is enhanced, the effects of casein kinase II-mediated phosphorylation on the individual steps of the catalytic cycle of Drosophila topoisomerase II were characterized. Phosphorylation did not affect reaction steps that preceded hydrolysis of the enzyme's high energy ATP cofactor. This included enzyme-DNA binding, pre-strand passage DNA cleavage/religation, the double-stranded DNA passage event, and post-strand passage DNA cleavage/religation. In contrast, the rate of topoisomerase II-mediated ATP hydrolysis was stimulated 2.7-fold following phosphorylation by casein kinase II. Since ATP hydrolysis is a prerequisite for enzyme turnover, it is concluded that phosphorylation modulates the overall catalytic activity of topoisomerase II by stimulating the enzyme's ATPase activity.     

Inhibition of eukaryotic topoisomerase II by ultraviolet-induced cyclobutane pyrimidine dimers.

The effects of short wave ultraviolet (UV)-induced DNA lesions on the catalytic activity of Drosophila melanogaster topoisomerase II were investigated. The presence of these photoproducts impaired the enzyme's ability to relax negatively supercoiled pBR322 plasmid molecules. As determined by DNA photolyase-catalyzed photoreactivation experiments, enzyme inhibition was due to the presence of cyclobutane pyrimidine dimers in the DNA. When 10-20 cyclobutane dimers were present per plasmid, the initial velocity of topoisomerase II-catalyzed DNA relaxation was inhibited approximately 50%. Decreased relaxation activity correlated with an inhibition of the DNA strand passage step of the enzyme's catalytic cycle. In contrast, UV-induced photoproducts did not alter the prestrand passage DNA cleavage/religation equilibrium of topoisomerase II either in the absence or presence of antineoplastic agents. Results of the present study demonstrate that the repair of cyclobutane pyrimidine dimers is important for the efficient catalytic function of topoisomerase II.     

Double-stranded DNA cleavage/religation reaction of eukaryotic topoisomerase II: evidence for a nicked DNA intermediate

The DNA cleavage reaction of eukaryotic topoisomerase II produces nicked DNA along with linear nucleic acid products. Therefore, relationships between the enzyme's DNA nicking and double-stranded cleavage reactions were determined. This was accomplished by altering the pH at which assays were performed. At pH 5.0 Drosophila melanogaster topoisomerase II generated predominantly (greater than 90%) single-stranded breaks in duplex DNA. With increasing pH, less single-stranded and more double-stranded cleavage was observed, regardless of the buffer or the divalent cation employed. As has been shown for double-stranded DNA cleavage, topoisomerase II was covalently bound to nicked DNA products, and enzyme-mediated single-stranded cleavage was salt reversible. Moreover, sites of single-stranded DNA breaks were identical with those mapped for double-stranded breaks. To further characterize the enzyme's cleavage mechanism, electron microscopy studies were performed. These experiments revealed that separate polypeptide chains were complexed with both ends of linear DNA molecules generated during cleavage reactions. Finally, by use of a novel religation assay [Osheroff, N., & Zechiedrich, E. L. (1987) Biochemistry 26, 4303-4309], it was shown that nicked DNA is an obligatory kinetic intermediate in the topoisomerase II mediated reunion of double-stranded breaks. Under the conditions employed, the apparent first-order rate constant for the religation of the first break was approximately 6-fold faster than that for the religation of the second break. The above results indicate that topoisomerase II carries out double-stranded DNA cleavage/religation by making two sequential single-stranded breaks in the nucleic acid backbone, each of which is mediated by a separate subunit of the homodimeric enzyme.     

Cross-resistance of an amsacrine-resistant human leukemia line to topoisomerase II reactive DNA intercalating agents. Evidence for two topoisomerase II directed drug actions.

HL-60/AMSA is a human leukemia cell line that is 50-100-fold more resistant than its drug-sensitive HL-60 parent line to the cytotoxic actions of the DNA intercalator amsacrine (m-AMSA). HL-60/AMSA topoisomerase II is also resistant to the inhibitory actions of m-AMSA. HL-60/AMSA cells and topoisomerase II are cross-resistant to anthracycline and ellipticine intercalators but relatively sensitive to the nonintercalating topoisomerase II reactive epipodophyllotoxin etoposide. We now demonstrate that HL-60/AMSA and its topoisomerase II are cross-resistant to the DNA intercalators mitoxantrone and amonafide, thus strongly indicating that HL-60/AMSA and its topoisomerase II are resistant to topoisomerase II reactive intercalators but not to nonintercalators. At high concentrations, mitoxantrone and amonafide were also found to inhibit their own, m-AMSA's, and etoposide's abilities to stabilize topoisomerase II-DNA complexes. This appears to be due to the ability of these concentrations of mitoxantrone and amonafide to inhibit topoisomerase II mediated DNA strand passage at a point in the topoisomerization cycle prior to the acquisition of the enzyme-DNA configuration that yields DNA cleavage and topoisomerase II-DNA cross-links. In addition, amonafide can inhibit the cytotoxic actions of m-AMSA and etoposide. Taken together, these results suggest that the cytotoxicity of m-AMSA and etoposide is initiated primarily by the stabilization of the topoisomerase II-DNA complex. Other topoisomerase II reactive drugs may inhibit the enzyme at other steps in the topoisomerization cycle, particularly at elevated concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mode of action of topoisomerase II-targeting agents at a specific DNA sequence. Uncoupling the DNA binding, cleavage and religation events.

Methods of uncoupling the DNA binding, cleavage and religation reactions of topoisomerase II were employed to investigate the influence of topoisomerase II-directed drugs on the individual steps in the enzyme's catalytic cycle. A special DNA substrate containing a major topoisomerase II interaction site, which can be cleaved by the enzyme in the absence of any concomitant religation, was used to examine the effect of topoisomerase II-directed agents upon the DNA cleavage reaction. The experiment demonstrated that the topoisomerase II targeting agent Ro 15-0216 stimulates the DNA cleavage reaction extensively, whereas the traditional topoisomerase II inhibitor, mAMSA, has only a minor effect on this reaction. Topoisomerase II trapped in the cleavage complexes can religate to the 3' hydroxyl end of another DNA strand. Using this religation assay, it was demonstrated that the major effect of mAMSA is an inhibition of the enzyme's religation reaction, whereas Ro 15-0216 has no effect on this reaction. Recently, considerable attention has been given to drugs preventing topoisomerase II from introducing DNA cleavages. In the present paper the initial non-covalent DNA binding reaction of topoisomerase II was investigated under conditions excluding enzyme-mediated DNA cleavage. This demonstrated that the anthracycline, aclarubicin, prevents topoisomerase II from performing its initial non-covalent DNA binding reaction and thereby abolishes the DNA cleavage reaction of the enzyme. The results presented here demonstrate that profound differences exist in the mode of action of different agents targeting topoisomerase II, and that the enzyme can be affected by such agents at both its DNA binding, cleavage and religation subreactions.     

Dissecting the cell-killing mechanism of the topoisomerase II-targeting drug ICRF-193

Topoisomerase II is an essential enzyme that is targeted by a number of clinically valuable anticancer drugs. One class referred to as topoisomerase II poisons works by increasing the cellular level of topoisomerase II-mediated DNA breaks, resulting in apoptosis. Another class of topoisomerase II-directed drugs, the bis-dioxopiperazines, stabilizes the conformation of the enzyme where it attains an inactive salt-stable closed clamp structure. Bis-dioxopiperazines, similar to topoisomerase II poisons, induce cell killing, but the underlying mechanism is presently unclear. In this study, we use three different biochemically well characterized human topoisomerase IIalpha mutant enzymes to dissect the catalytic requirements needed for the enzyme to cause dominant sensitivity in yeast to the bis-dioxopirazine ICRF-193 and the topoisomerase II poison m-AMSA. We find that the clamp-closing activity, the DNA cleavage activity, and even both activities together are insufficient for topoisomerase II to cause dominant sensitivity to ICRF-193 in yeast. Rather, the strand passage event per se is an absolute requirement, most probably because this involves a simultaneous interaction of the enzyme with two DNA segments. Furthermore, we show that the ability of human topoisomerase IIalpha to cause dominant sensitivity to m-AMSA in yeast does not depend on clamp closure or strand passage but is directly related to the capability of the enzyme to respond to m-AMSA with increased DNA cleavage complex formation.     

Studies of the topoisomerase II-mediated cleavage and religation reactions by use of a suicidal double-stranded DNA substrate.

The cleavage and religation reactions of eukaryotic topoisomerase II were studied by use of a 5'-recessed DNA substrate containing a strong recognition sequence for the enzyme. Cleavage of the DNA substrate was suicidal, that is the enzyme was unable to religate the cleaved DNA due to a release of DNA 5' to the cleavage position. With this substrate cleavage products accumulated with time in the absence of protein-denaturing agents, and the cleavage reaction was not reversible with salt. The suicide cleavage complexes contained a kinetically competent topoisomerase II enzyme as determined by the enzyme's ability to perform intermolecular ligation of the cleaved DNA to a free 3'-hydroxyl end on another DNA strand. The efficiency of the religation reaction depended on the ability of the religation substrate to base pair to the DNA in the cleaved enzyme-DNA complex. Higher levels of religation were obtained with dinucleotides than with long DNA substrates. Mononucleotides also were efficiently religated, indicating an ability of the enzyme to mediate religation without making contacts to a long stretch of nucleotides 5' to the cleavage position.     

Characterization of an amsacrine-resistant line of human leukemia cells. Evidence for a drug-resistant form of topoisomerase II

HL-60/AMSA is a human leukemia cell line that is 100 times more resistant to the cytotoxic actions of the antineoplastic, topoisomerase II-reactive DNA intercalating acridine derivative amsacrine (m-AMSA) than is its parent HL-60 line. HL-60/AMSA cells are minimally resistant to etoposide, a topoisomerase II-reactive drug that does not intercalate. Previously we showed that HL-60 topoisomerase II activity in cells, nuclei, or nuclear extracts was sensitive to m-AMSA and etoposide, while HL-60/AMSA topoisomerase II was resistant to m-AMSA but sensitive to etoposide. Now we show that purified topoisomerase II from the two cell lines exhibits the same drug sensitivity or resistance as that in the nuclear extracts although the magnitude of the m-AMSA resistance of HL-60/AMSA topoisomerase II in vitro is not as great as the resistance of the intact HL-60/AMSA cells. In addition HL-60/AMSA cells are cross-resistant to topoisomerase II-reactive intercalators from the anthracycline and ellipticine families and the pattern of sensitivity or resistance to the cytotoxic actions of the various topoisomerase II-reactive drugs is paralleled by topoisomerase II-reactive drug-induced DNA cleavage and protein cross-link production in cells and the production of drug-induced, topoisomerase II-mediated DNA cleavage and protein cross-linking in isolated biochemical systems. In addition to its lowered sensitivity to intercalators, HL-60/AMSA differed from HL-60 in 1) the susceptibility of its topoisomerase II to stimulation of DNA topoisomerase II complex formation by ATP, 2) the catalytic activity of its topoisomerase II in an ionic environment chosen to reproduce the environment found within the living cell, and 3) the observed restriction enzyme pattern on a Southern blot probed with a cDNA for human topoisomerase II. These data indicate that an m-AMSA-resistant form of topoisomerase II contributes to the resistance of HL-60/AMSA to m-AMSA and to other topoisomerase II-reactive DNA intercalating agents. The drug resistance is associated with additional biochemical and molecular alterations that may be important determinants of cellular sensitivity or resistance to topoisomerase II-reactive drugs.     

Eukaryotic topoisomerase II. Characterization of enzyme turnover

While the binding of adenyl-5'-yl imidodiphosphate (App(NH)p) to Drosophila melanogaster topoisomerase II induces a double-stranded DNA passage reaction, its nonhydrolyzable beta,gamma-imidodiphosphate bond prevents enzyme turnover (Osheroff, N., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9536-9543). Therefore, this ATP analog was used to characterize the interactions between Drosophila topoisomerase II and DNA which occur after DNA strand passage but before enzyme turnover. In the presence of App(NH)p, a stable post-strand passage topoisomerase II-nucleic acid complex is formed when circular DNA substrates are employed. Although noncovalent in nature, these complexes are resistant to increases in ionic strength and show less than 5% dissociation under salt concentrations (greater than 500 mM) that disrupt 95% of the enzyme-DNA interactions formed in the absence of App(NH)p or under a variety of other conditions that do not support DNA strand passage. These results strongly suggest that the process of enzyme turnover not only regenerates the active conformation of topoisomerase II but also confers upon the enzyme the ability to disengage from its nucleic acid product. Experiments with linear DNA molecules indicate that after strand passage has taken place, topoisomerase II may be able to travel along its DNA substrate by a linear diffusion process that is independent of enzyme turnover. Further studies demonstrate that the regeneration of the enzyme's catalytic center does not require enzyme turnover, since topoisomerase II can cleave double-stranded DNA substrates after strand passage has taken place. Finally, while the 2'-OH and 3'-OH of ATP are important for its interaction with Drosophila topoisomerase II, neither are required for turnover.     

Strand specificity of the topoisomerase II mediated double-stranded DNA cleavage reaction

The strand specificity of topoisomerase II mediated DNA cleavage was analyzed at the nucleotide level by characterizing the enzyme's interaction with a strong DNA recognition site. This site was isolated from the promoter region of the extrachromosomal rRNA genes of Tetrahymena thermophila and was recognized by type II topoisomerases from a variety of phylogenetically diverse eukaryotic organisms, including Drosophila, Tetrahymena, and calf thymus. When incubated with this site, topoisomerase II was found to introduce single-stranded breaks (i.e., nicks) in addition to double-stranded breaks in the nucleic acid backbone. Although the nucleotide position of cleavage on both the noncoding and coding strands of the rDNA remained unchanged, the relative ratios of single- and double-stranded DNA breaks could be varied by altering reaction conditions. Under all conditions which promoted topoisomerase II mediated DNA nicking, the enzyme displayed a 3-10-fold specificity for cleavage at the noncoding strand of its recognition site. To determine whether this specificity of topoisomerase II was due to a faster forward rate of cleavage of the noncoding strand or a slower rate of its religation, a DNA religation assay was performed. Results indicated that both the noncoding and coding strands were religated by the enzyme at approximately the same rate. Therefore, the DNA strand preference of topoisomerase II appears to be embodied in the enzyme's forward cleavage reaction.     

A role for the passage helix in the DNA cleavage reaction of eukaryotic topoisomerase II. A two-site model for enzyme-mediated DNA cleavage.

Eukaryotic topoisomerase II is capable of binding two separate nucleic acid helices prior to its DNA cleavage and strand passage events (Zechiedrich, E. L., and Osheroff, N (1990) EMBO J. 9, 4555-4562). Presumably, one of these helices represents the helix that the enzyme cleaves (i.e. cleavage helix), and the other represents the helix that it passes (i.e. passage helix) through the break in the nucleic acid backbone. To determine whether the passage helix is required for reaction steps that precede the enzyme's DNA strand passage event, interactions between Drosophila melanogaster topoisomerase II and a short double-stranded oligonucleotide were assessed. These studies employed a 40-mer that contained a specific recognition/cleavage site for the enzyme. The sigmoidal DNA concentration dependence that was observed for cleavage of the 40-mer indicated that topoisomerase II had to interact with more than a single oligonucleotide in order for cleavage to take place. Despite this requirement, results of enzyme DNA binding experiments indicated no binding cooperativity for the 40-mer. These findings strongly suggest a two-site model for topoisomerase II action in which the passage and the cleavage helices bind to the enzyme independently, but the passage helix must be present for efficient topoisomerase II-mediated DNA cleavage to occur.     

DNA topoisomerases as targets for the anticancer drug TAS-103: primary cellular target and DNA cleavage enhancement

TAS-103 is a novel antineoplastic agent that is active against in vivo tumor models [Utsugi, T., et al. (1997) Jpn. J. Cancer Res. 88, 992-1002]. This drug is believed to be a dual topoisomerase I/II-targeted agent, because it enhances both topoisomerase I- and topoisomerase II-mediated DNA cleavage in treated cells. However, the relative importance of these two enzymes for the cytotoxic actions of TAS-103 is not known. Therefore, the primary cellular target of the drug and its mode of action were determined. TAS-103 stimulated DNA cleavage mediated by mammalian topoisomerase I and human topoisomerase IIalpha and beta in vitro. The drug was less active than camptothecin against the type I enzyme but was equipotent to etoposide against topoisomerase IIalpha. A yeast genetic system that allowed manipulation of topoisomerase activity and drug sensitivity was used to determine the contributions of topoisomerase I and II to drug cytotoxicity. Results indicate that topoisomerase II is the primary cellular target of TAS-103. In addition, TAS-103 binds to human topoisomerase IIalpha in the absence of DNA, suggesting that enzyme-drug interactions play a role in formation of the ternary topoisomerase II.drug.DNA complex. TAS-103 induced topoisomerase II-mediated DNA cleavage at sites similar to those observed in the presence of etoposide. Like etoposide, it enhanced cleavage primarily by inhibiting the religation reaction of the enzyme. Based on these findings, it is suggested that TAS-103 be classified as a topoisomerase II-targeted drug.     

Quinolones inhibit DNA religation mediated by Staphylococcus aureus topoisomerase IV. Changes in drug mechanism across evolutionary boundaries

Quinolones are the most active oral antibacterials in clinical use and act by increasing DNA cleavage mediated by prokaryotic type II topoisomerases. Although topoisomerase IV appears to be the primary cytotoxic target for most quinolones in Gram-positive bacteria, interactions between the enzyme and these drugs are poorly understood. Therefore, the effects of ciprofloxacin on the DNA cleavage and religation reactions of Staphylococcus aureus topoisomerase IV were characterized. Ciprofloxacin doubled DNA scission at 150 nM drug and increased cleavage approximately 9-fold at 5 microM. Furthermore, it dramatically inhibited rates of DNA religation mediated by S. aureus topoisomerase IV. This inhibition of religation is in marked contrast to the effects of antineoplastic quinolones on eukaryotic topoisomerase II, and suggests that the mechanistic basis for quinolone action against type II topoisomerases has not been maintained across evolutionary boundaries. The apparent change in quinolone mechanism was not caused by an overt difference in the drug interaction domain on topoisomerase IV. Therefore, we propose that the mechanistic basis for quinolone action is regulated by subtle changes in drug orientation within the enzyme.drug.DNA ternary complex rather than gross differences in the site of drug binding.     

Numerical Analysis of Etoposide Induced DNA Breaks

Background

Etoposide is a cancer drug that induces strand breaks in cellular DNA by inhibiting topoisomerase II (topoII) religation of cleaved DNA molecules. Although DNA cleavage by topoisomerase II always produces topoisomerase II-linked DNA double-strand breaks (DSBs), the action of etoposide also results in single-strand breaks (SSBs), since religation of the two strands are independently inhibited by etoposide. In addition, recent studies indicate that topoisomerase II-linked DSBs remain undetected unless topoisomerase II is removed to produce free DSBs.

Methodology/Principal Findings

To examine etoposide-induced DNA damage in more detail we compared the relative amount of SSBs and DSBs, survival and H2AX phosphorylation in cells treated with etoposide or calicheamicin, a drug that produces free DSBs and SSBs. With this combination of methods we found that only 3% of the DNA strand breaks induced by etoposide were DSBs. By comparing the level of DSBs, H2AX phosphorylation and toxicity induced by etoposide and calicheamicin, we found that only 10% of etoposide-induced DSBs resulted in histone H2AX phosphorylation and toxicity. There was a close match between toxicity and histone H2AX phosphorylation for calicheamicin and etoposide suggesting that the few etoposide-induced DSBs that activated H2AX phosphorylation were responsible for toxicity.

Conclusions/Significance

These results show that only 0.3% of all strand breaks produced by etoposide activate H2AX phosphorylation and suggests that over 99% of the etoposide induced DNA damage does not contribute to its toxicity.     

Cobalt enhances DNA cleavage mediated by human topoisomerase II alpha in vitro and in cultured cells

Although cobalt is an essential trace element for humans, the metal is genotoxic and mutagenic at higher concentrations. Treatment of cells with cobalt generates DNA strand breaks and covalent protein-DNA complexes. However, the basis for these effects is not well understood. Since the toxic events induced by cobalt resemble those of topoisomerase II poisons, the effect of the metal on human topoisomerase IIalpha was examined. The level of enzyme-mediated DNA scission increased 6-13-fold when cobalt(II) replaced magnesium(II) in cleavage reactions. Cobalt(II) stimulated cleavage at all DNA sites observed in the presence of magnesium(II), and the enzyme cut DNA at several "cobalt-specific" sites. The increased level of DNA cleavage in the presence of cobalt(II) was partially due to a decrease in the rate of enzyme-mediated religation. Topoisomerase IIalpha retained many of its catalytic properties in reactions that included cobalt(II), including sensitivity to the anticancer drug etoposide and the ability to relax and decatenate DNA. Finally, cobalt(II) stimulated topoisomerase IIalpha-mediated DNA cleavage in the presence of magnesium(II) in purified systems and in human MCF-7 cells. These findings demonstrate that cobalt(II) is a topoisomerase II poison in vitro and in cultured cells and suggest that at least some of the genotoxic effects of the metal are mediated through topoisomerase IIalpha.     

In vivo mapping of DNA topoisomerase II-specific cleavage sites on SV40 chromatin

The antitumor drug, m-AMSA (4'-(9-acridinylamino)-methanesulfon-m-anisidide), is known to interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by blocking the enzyme-DNA complex in its putative cleavable state. Treatment of SV40 virus infected monkey cells with m-AMSA resulted in both single- and double-stranded breaks on SV40 viral chromatin. These strand breaks are unusual because they are covalently associated with protein. Immunoprecipitation results suggest that the covalently linked protein is DNA topoisomerase II. These results are consistent with the proposal that the drug action in vivo involves the stabilization of a cleavable complex between topoisomerase II and DNA in chromatin. Mapping of these double-stranded breaks on SV40 viral DNA revealed multiple topoisomerase II cleavage sites. A major topoisomerase II cleavage site was preferentially induced during late infection and was mapped in the DNAase I hypersensitive region of SV40 chromatin.     

Intrinsic intermolecular DNA ligation activity of eukaryotic topoisomerase II. Potential roles in recombination.

Drosophila melanogaster topoisomerase II is capable of joining phi X174 (+) strand DNA that it has cleaved to duplex oligonucleotide acceptor molecules by an intermolecular ligation reaction (Gale, K. C. and Osheroff, N. (1990) Biochemistry 29, 9538-9545). In order to investigate potential mechanisms for topoisomerase II-mediated DNA recombination, this intrinsic enzyme activity was further characterized. Intermolecular DNA ligation proceeded in a time-dependent fashion and was concentration-dependent with respect to oligonucleotide. The covalent linkage between phi X174 (+) strand DNA and acceptor molecules was confirmed by Southern analysis and alkaline gel electrophoresis. Topoisomerase II-mediated intermolecular DNA ligation required the oligonucleotide to contain a 3'-OH terminus. Moreover, the reaction was dependent on the presence of a divalent cation, was inhibited by salt, and was not affected by the presence of ATP. The enzyme was capable of ligating phi X174 (+) strand DNA to double-stranded oligonucleotides that contained 5'-overhang, 3'-overhand, or blunt ends. Single-stranded, nicked, or gapped oligonucleotides also could be used as acceptor molecules. These results demonstrate that the type II enzyme has an intrinsic ability to mediate illegitimate DNA recombination in vitro and suggests possible roles for topoisomerase II in nucleic acid recombination in vivo.     

The production of topoisomerase II-mediated DNA cleavage in human leukemia cells predicts their susceptibility to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)

Protein-associated DNA cleavage is produced in mammalian cells treated with active antileukemic DNA intercalating agents such as 4'(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). We have examined the ability of m-AMSA to produce DNA cleavage in 3 human myeloid leukemic cell lines with different sensitivities to the cytotoxic actions of m-AMSA to see if the magnitude of DNA cleavage correlated with the degree of m-AMSA sensitivity. DNA alkaline elution was used to quantify DNA cleavage. The amount of m-AMSA-induced DNA cleavage in the two lines sensitive to m-AMSA was 1-2 orders of magnitude greater than that in an m-AMSA-resistant leukemic line. The m-AMSA resistant line had been developed by prolonged exposure of one of the sensitive lines to m-AMSA. This finding was not secondary to a decreased uptake of m-AMSA in the resistant cell line. m-AMSA treatment of the nuclei isolated from the three lines produced DNA cleavage frequencies comparable to the cleavage frequencies produced by m-AMSA treatment of the whole cells from which the nuclei were isolated. The DNA cleaving ability stimulated by m-AMSA is thought to be mediated by drug-induced effects on topoisomerase II, a nuclear enzyme that mediates alterations in DNA conformation. Alterations in the manner in which this enzyme interacts with antineoplastic agents may explain the emergence of resistant cells following initially successful chemotherapy.