Separation of bisdioxopiperazine- and vanadate resistance in topoisomerase II

Bisdioxopiperazines are inhibitors of topoisomerase II trapping this protein as a closed clamp on DNA with concomitant inhibition of its ATPase activity. Here, we analyse the effects of N-terminal mutations identified in bisdioxopiperazine-resistant cells on ATP hydrolysis by this enzyme. We present data consistent with bisdioxopiperazine resistance arising by two different mechanisms; one involving reduced stability of the N-terminal clamp (the N-gate) and one involving reduced affinity for bisdioxopiperazines. Vanadate is a general inhibitor of type P ATPases and has recently been demonstrated to lock topoisomerase II as a salt-stable closed clamp on DNA analogous to the bisdioxopiperazines. We show that a R162K mutation in human topoisomerase II alpha renders this enzyme highly resistant towards vanadate while having little effect on bisdioxopiperazine sensitivity. The implications of these findings for the mechanism of action of bisdioxopiperazines versus vanadate with topoisomerase II are discussed.     

Interaction of human DNA topoisomerase II alpha with DNA: quantification by surface plasmon resonance

DNA topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double-stranded DNA segment and transporting it through another duplex. Surface plasmon resonance (SPR) was used to characterize interactions of human topoisomerase II alpha with different topological forms of DNA. Using a linear fragment of pUC18 DNA, the equilibrium binding constant of topoisomerase II alpha was determined to be 0.16 nM. The affinity was not affected by the absence of ATP or the presence of the bisdioxopiperazine catalytic inhibitor ICRF-187. Besides, similar affinities were found for several bisdioxopiperazine-resistant mutant enzymes. These results suggest that the mechanism of topoisomerase II alpha inhibition by ICRF-187 and its resistance does not directly involve the interaction of DNA with the enzyme. SPR was also adapted to measure levels of the closed clamp form of topoisomerase II present on DNA. As expected, a stable closed clamp form of the enzyme was detectable on circular DNA but not on linear DNA. Detection of the closed clamp required the presence of ATP and a bisdioxopiperazine, or a non-hydrolyzable analogue of ATP. In the presence of ATP and ICRF-187, several bisdioxopiperazine-resistant mutant enzymes failed to form detectable levels of stable closed clamp. Interestingly, a mutant of human topoisomerase II alpha with an altered active site tyrosine showed lower levels of closed clamp formation. In conclusion, SPR is able to (1) determine the kinetics of topoisomerase II with its DNA substrate and (2) quantify the enzyme's closed clamp formation under varying circumstances.     

N-terminal and core-domain random mutations in human topoisomerase II α conferring bisdioxopiperazine resistance

Random mutagenesis of human topoisomerase II α cDNA followed by functional expression in yeast cells lacking endogenous topoisomerase II activity in the presence of ICRF-187, identified five functional mutations conferring cellular bisdioxopiperazine resistance. The mutations L169F, G551S, P592L, D645N, and T996L confer >37, 37, 18, 14, and 19 fold resistance towards ICRF-187 in a 24 h clonogenic assay, respectively. Purified recombinant L169F protein is highly resistant towards catalytic inhibition by ICRF-187 in vitro while G551S, D645N, and T996L proteins are not. This demonstrates that cellular bisdioxopiperazine resistance can result from at least two classes of mutations in topoisomerase II; one class renders the protein non-responsive to bisdioxopiperazine compounds, while an other class does not appear to affect the catalytic sensitivity towards these drugs. In addition, our results indicate that different protein domains are involved in mediating the effect of bisdioxopiperazine compounds.     

Synthesis and characterization of the biological activity of the cisplatin analogs, cis-PtCl(2)(dexrazoxane) and cis-PtCl(2)(levrazoxane), of the topoisomerase II inhibitors dexrazoxane (ICRF-187) and levrazoxane (ICRF-186)

The individual stereoisomers cis-PtCl(2)(dexrazoxane) and cis-PtCl(2)(levrazoxane) were synthesized and their structures were determined by X-ray crystallography. Dexrazoxane and levrazoxane inhibit cell growth because they are strong catalytic inhibitors of DNA topoisomerase II, whereas cisplatin acts through the formation of DNA cross-links. It was hypothesized that platinum(II) complexes of dexrazoxane and levrazoxane would retain both activities and yield drugs with a dual mode of action. Both cis-PtCl(2)(dexrazoxane) and cis-PtCl(2)(levrazoxane) inhibited Chinese hamster ovary cell growth, but more weakly than dexrazoxane and levrazoxane did. Based on their weak topoisomerase II inhibitory activity, it was concluded that these compounds did not inhibit cell growth by targeting topoisomerase II. A comparison of the conformation of cis-PtCl(2)(dexrazoxane) to that of dexrazoxane bound to the dimer interface of topoisomerase II showed that the highly constrained cis-PtCl(2)(dexrazoxane) was in a highly unfavorable conformation for binding. Neither of the platinum complexes were able to cross-link DNA. Thus the cell growth inhibitory activity of these complexes was also not likely due to any cisplatin-type cross-linking activity.     

Structural Insights into the Quinolone Resistance Mechanism of Mycobacterium tuberculosis DNA Gyrase

Mycobacterium tuberculosis DNA gyrase, an indispensable nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and is hence the sole target for quinolone action, a crucial drug active against multidrug-resistant tuberculosis. To understand at an atomic level the quinolone resistance mechanism, which emerges in extensively drug resistant tuberculosis, we performed combined functional, biophysical and structural studies of the two individual domains constituting the catalytic DNA gyrase reaction core, namely the Toprim and the breakage-reunion domains. This allowed us to produce a model of the catalytic reaction core in complex with DNA and a quinolone molecule, identifying original mechanistic properties of quinolone binding and clarifying the relationships between amino acid mutations and resistance phenotype of M. tuberculosis DNA gyrase. These results are compatible with our previous studies on quinolone resistance. Interestingly, the structure of the entire breakage-reunion domain revealed a new interaction, in which the Quinolone-Binding Pocket (QBP) is blocked by the N-terminal helix of a symmetry-related molecule. This interaction provides useful starting points for designing peptide based inhibitors that target DNA gyrase to prevent its binding to DNA.     

Self-association and DNA binding properties of the human topoisomerase IIA alpha2HTH module

The eukaryotic topoisomerase II is an ubiquitous nuclear enzyme involved in vital cellular functions. It is also the target for some of the most active anticancer drugs. In the various crystal structures of yeast topoisomerase II, the 701-748 segment homologous to the human topoisomerase II alpha 724-771 segment folds into a compact alpha(2)beta(1)alpha(3)talpha(4) conformation, hereafter termed alpha(2)HTH module (helix turn helix (HTH), alpha(3)talpha(4)). The crystal structure of gyrase A has suggested a model wherein HTH is involved in both the enzyme dimerization and the binding to DNA. These two properties were investigated in solution, using the recombinant alpha(2)HTH module of human topoisomerase II alpha and its synthetic components HTH, alpha(4), alpha(3) and turn. The homology-based structure model of human alpha(2)HTH superposed that of yeast in the crystal structure with a rmsd of 1.03 A. Circular dichroism spectra showed that the helical content of human alpha(2)HTH in solution is similar to that of its counterpart within yeast topoisomerase II in the solid state. The chemical cross-linking data indicated that alpha(2)HTH self-associated into dimers while gel mobility shift assays and anisotropy fluorescence titrations demonstrated that alpha(2)HTH, HTH and alpha(4), but not alpha(3), bind efficiently to DNA (dissociation constants of 3.10(-7) M for alpha(2)HTH and alpha(4), of 3.10(-6) M for HTH and of only 1.10(-5) M for alpha(3)). Correlatively, alpha(2)HTH, alpha(4) and HTH, but not alpha(3), were able to inhibit topoisomerase II in DNA relaxation assays, stipulating that alpha(4) is the DNA recognition helix. All suggests that the alpha(2)HTH module once separated from the whole protein conserves a compact conformation, integral to specific dimerization and DNA recognition. The module may thus be used for the search of drugs efficient in hindering topoisomerase II dimerization or binding to DNA.     

Human small cell lung cancer NYH cells resistant to the bisdioxopiperazine ICRF-187 exhibit a functional dominant Tyr165Ser mutation in the Walker A ATP binding site of topoisomerase II alpha

Bisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme's Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme.     

DNA-drug recognition and effects on topoisomerase II-mediated cytotoxicity. A three-mode binding model for ellipticine derivatives.

Cytotoxic effects and topoisomerase II-mediated DNA breaks induced in vitro by ellipticine derivatives were examined in connection with 1H NMR and circular dichroism (CD) studies on molecular structures and interactions of drugs with DNA. The compounds included four 9-hydroxyellipticine and two 7-hydroxyisoellipticine derivatives. Structure-activity relationships indicated that a change in nitrogen atom position in the pyridinic ring greatly affected drug effects both on topoisomerase II action and cytotoxicity to L1210 cells. The four 9-hydroxyellipticine derivatives yielded bell-shaped curves in in vitro topoisomerase II-mediated DNA break assays, whereas the two 7-hydroxyisoellipticine derivatives demonstrated an almost linear increase at the same concentration (0-10 microM). In both cases, the intensity of cleavage was modulated by the position and the degree of methylation on the pyridinic ring, and results were correlated with cytotoxic activity expressed as the in vitro ID50 values for L1210 leukemia cells. 1H NMR experiments performed on free drug molecules in solution revealed that the two protons (alpha and beta) contiguous to the biologically important hydroxy group were sensitive to changes in electron distribution produced by the distant chemical modifications and methylations of the pyridinic ring. A linear relationship was observed between the differences in chemical shifts of alpha and beta protons (delta delta alpha-beta) versus ID50 values. CD experiments indicated that, at weak ionic strength I = 0.02 and at pH 7, drugs interact with the poly[d(A-T)] duplex according to a "three-mode binding model" which is governed by the drug structure and the drug to DNA ratio. The intercalation mode was related to the induction of topoisomerase II-mediated DNA cleavage, while the external binding mode consecutive to intercalation was related to cleavage suppression. These two modes concerned the good intercalators 9-hydroxyellipticines. The third was found for the weak intercalators 7-hydroxyisoellipticines and was characterized by self-stacked molecules bound "outside" DNA, presumably in the minor groove. Ligands either could be intercalated partially or linked at the edge of bases with a small number of molecules filling intercalation sites, for the second alternative. In addition to having different binding modes, 9-hydroxyellipticines were better inducers of DNA distortions than 7-hydroxyisoellipticines. The incidence of the drug binding modes on DNA-topoisomerase II recognition was discussed in connection with the in vitro cytotoxic activity exhibited by the drugs.     

A two-drug model for etoposide action against human topoisomerase IIalpha

The widely used anticancer drug etoposide kills cells by increasing levels of topoisomerase II-mediated DNA breaks. While it is known that the drug acts by inhibiting the ability of topoisomerase II to ligate cleaved DNA molecules, the precise mechanism by which it accomplishes this action is not well understood. Because there are two scissile bonds per enzyme-mediated double-stranded DNA break, it has been assumed that there are two sites for etoposide in every cleavage complex. However, it is not known whether the action of etoposide at only one scissile bond is sufficient to stabilize a double-stranded DNA break or whether both drug sites need to be occupied. An oligonucleotide system was utilized to address this important issue. Results of DNA cleavage and ligation assays support a two-drug model for the action of etoposide against human topoisomerase IIalpha. This model postulates that drug interactions at both scissile bonds are required in order to increase enzyme-mediated double-stranded DNA breaks. Etoposide actions at either of the two scissile bonds appear to be independent of one another, with each individual drug molecule stabilizing a strand-specific nick rather than a double-stranded DNA break. This finding suggests (at least in the presence of drug) that there is little or no communication between the two promoter active sites of topoisomerase II. The two-drug model has implications for cancer chemotherapy, the cellular processing of etoposide-stabilized enzyme-DNA cleavage complexes, and the catalytic mechanism of eukaryotic topoisomerase II.     

Evidence for a common mechanism of action for antitumor and antibacterial agents that inhibit type II DNA topoisomerases

Numerous antitumor and antibacterial agents inhibit type II DNA topoisomerases, yielding, in each case, a complex of enzyme covalently bound to cleaved DNA. We are investigating the mechanism of inhibitor action by using the type II DNA topoisomerase of bacteriophage T4 as a model. The T4 topoisomerase is the target of antitumor agent 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA) in T4-infected Escherichia coli. Two m-AMSA-resistant phage strains were previously isolated, one with a point mutation in topoisomerase subunit gene 39 and the other with a point mutation in topoisomerase subunit gene 52. We report here that the wild-type T4 topoisomerase is inhibited by six additional antitumor agents that also inhibit the mammalian type II topoisomerase: ellipticine, 9-OH-ellipticine, 2-me-9-OH-ellipticinium acetate, mitoxantrone diacetate, teniposide, and etoposide. Further, one or both of the m-AMSA-resistance mutations alters the enzyme sensitivity to each of these agents, conferring either cross-resistance or enhanced sensitivity. Finally, the gene 39 mutation confers on T4 topoisomerase a DNA gyrase-like sensitivity to the gyrase inhibitor oxolinic acid, thus establishing a direct link between the mechanism of action of the anti-bacterial quinolones and that of the antitumor agents. These results strongly suggest that diverse inhibitors of type II topoisomerases share a common binding site and a common mechanism of action, both of which are apparently conserved in the evolution of the type II DNA topoisomerases. Alterations in DNA cleavage site specificity caused by either the inhibitors or the m-AMSA-resistance mutations favor the proposal that the inhibitor binding site is composed of both protein and DNA.     

A human topoisomerase II alpha heterodimer with only one ATP binding site can go through successive catalytic cycles

Eukaryotic DNA topoisomerase II is a dimeric nuclear enzyme essential for DNA metabolism and chromosome dynamics. It changes the topology of DNA by coupling binding and hydrolysis of two ATP molecules to the transport of one DNA duplex through a temporary break introduced in another. During this process the structurally and functionally complex enzyme passes through a cascade of conformational changes, which requires intra- and intersubunit communication. To study the importance of ATP binding and hydrolysis in relation to DNA strand transfer, we have purified and characterized a human topoisomerase II alpha heterodimer with only one ATP binding site. The heterodimer was able to relax supercoiled DNA, although less efficiently than the wild type enzyme. It furthermore possessed a functional N-terminal clamp and was sensitive to ICRF-187. This demonstrates that human topoisomerase II alpha can pass through all the conformations required for DNA strand passage and enzyme resetting with binding and hydrolysis of only one ATP. However, the heterodimer lacked the normal stimulatory effect of DNA on ATP binding and hydrolysis as well as the stimulatory effect of ATP on DNA cleavage. The results can be explained in a model, where efficient catalysis requires an extensive communication between the second ATP and the DNA segment to be cleaved.     

Mutations of human topoisomerase II alpha affecting multidrug resistance and sensitivity.

Two mutations, R450Q and P803S, in the coding region of the human topoisomerase II alpha gene have been identified in the atypical multidrug resistant (at-MDR) cell line, CEM/VM-1, which exhibits resistance to many structurally diverse topoisomerase II-targeting antitumor drugs such as VM-26, doxorubicin, m-AMSA, and mitoxantrone. The R450Q mutation mapped in the ATP utilization domain, while the P803S mutation mapped in the vicinity of the active site tyrosine of human topoisomerase II alpha. However, the roles of these two mutations in conferring multidrug resistance are unclear. To study the roles of these two mutations in conferring multidrug resistance, we have characterized the recombinant human DNA topoisomerase II alpha containing either single or double mutations. We show that both R450Q and P803S mutations confer resistance in the absence of ATP. However, in the presence of ATP, the R450Q, but not the P803S, mutation can confer multidrug resistance. The R450Q enzyme was shown to exhibit impaired ATP utilization both for enzyme catalysis and for its ability to form the circular protein clamp. Interestingly, an unrelated mutation, G437E, which is also located in the same domain as the R450Q mutation, exhibited multidrug hypersensitivity in the absence of ATP. However, in the presence of ATP, the G437E enzyme is only minimally hypersensitive to various topoisomerase II drugs. In contrast to the R450Q enzyme, the G437E enzyme exhibited enhanced ATP utilization for enzyme catalysis. In the aggregate, these results support the notion that the multidrug resistance and sensitivity of these mutant enzymes are due to a specific defect in ATP utilization during enzyme catalysis.     

DNA topoisomerases as targets for the anticancer drug TAS-103: DNA interactions and topoisomerase catalytic inhibition

TAS-103 is a novel anticancer drug that kills cells by increasing levels of DNA cleavage mediated by topoisomerase II. While most drugs that stimulate topoisomerase II-mediated DNA scission (i.e., topoisomerase II poisons) also inhibit the catalytic activity of the enzyme, they typically do so only at concentrations above the clinical range. TAS-103 is unusual in that it reportedly inhibits the catalytic activity of both topoisomerase I and II and does so at physiologically relevant concentrations [Utsugi, T., et al. (1997) Jpn. J. Cancer Res. 88, 992-1002]. Without a topoisomerase activity to relieve accumulating torsional stress, the DNA tracking systems that promote the action of TAS-103 as a topoisomerase II poison would be undermined. Therefore, the effects of TAS-103 on the catalytic activity of topoisomerase I and II were characterized. DNA binding and unwinding assays indicate that the drug intercalates into DNA with an apparent dissociation constant of approximately 2.2 microM. Furthermore, DNA strand passage assays with mammalian topoisomerase I indicate that TAS-103 does not inhibit the catalytic activity of the type I enzyme. Rather, the previously reported inhibition of topoisomerase I-catalyzed DNA relaxation results from a drug-induced alteration in the apparent topology of the nucleic acid substrate. TAS-103 does inhibit the catalytic activity of human topoisomerase IIalpha, apparently by blocking the DNA religation reaction of the enzyme. The lack of inhibition of topoisomerase I catalytic activity by TAS-103 explains how the drug is able to function as a topoisomerase II poison in treated cells.     

Interactions between the etoposide derivative F14512 and human type II topoisomerases: implications for the C4 spermine moiety in promoting enzyme-mediated DNA cleavage

F14512 is a novel etoposide derivative that contains a spermine in place of the C4 glycosidic moiety. The drug was designed to exploit the polyamine transport system that is upregulated in some cancers. However, a preliminary study suggests that it is also a more efficacious topoisomerase II poison than etoposide [Barret et al. (2008) Cancer Res. 68, 9845-9853]. Therefore, we undertook a more complete study of the actions of F14512 against human type II topoisomerases. As determined by saturation transfer difference (1)H NMR spectroscopy, contacts between F14512 and human topoisomerase IIα in the binary enzyme-drug complex are similar to those of etoposide. Although the spermine of F14512 does not interact with the enzyme, it converts the drug to a DNA binder [Barret et al. (2008)]. Consequently, the influence of the C4 spermine on drug activity was assessed. F14512 is a highly active topoisomerase II poison and stimulates DNA cleavage mediated by human topoisomerase IIα or topoisomerase IIβ. The drug is more potent and efficacious than etoposide or TOP-53, an etoposide derivative that contains a C4 aminoalkyl group that strengthens drug-enzyme binding. Unlike the other drugs, F14512 maintains robust activity in the absence of ATP. The enhanced activity of F14512 correlates with a tighter binding and an increased stability of the ternary topoisomerase II-drug-DNA complex. The spermine-drug core linkage is critical for these attributes. These findings demonstrate the utility of a C4 DNA binding group and provide a rational basis for the development of novel and more active etoposide-based topoisomerase II poisons.     

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.     

Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly

Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drug-resistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase IIbeta. One can prevent the interaction by introducing a C(11)-to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase IIalpha/beta in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase IIalpha and IIbeta antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases IIalpha/beta in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.     

Modulation of drug sensitivity in yeast cells by the ATP-binding domain of human DNA topoisomerase IIalpha

Epipodophyllotoxins are effective antitumour drugs that trap eukaryotic DNA topoisomerase II in a covalent complex with DNA. Based on DNA cleavage assays, the mode of interaction of these drugs was proposed to involve amino acid residues of the catalytic site. An in vitro binding study, however, revealed two potential binding sites for etoposide within human DNA topoisomerase IIα (htopoIIα), one in the catalytic core of the enzyme and one in the ATP-binding N-terminal domain. Here we have tested how N-terminal mutations that reduce the affinity of the site for etoposide or ATP affect the sensitivity of yeast cells to etoposide. Surprisingly, when introduced into full-length enzymes, mutations that lower the drug binding capacity of the N-terminal domain in vitro render yeast more sensitive to epipodophyllotoxins. Consistently, when the htopoIIα N-terminal domain alone is overexpressed in the presence of yeast topoII, cells become more resistant to etoposide. Point mutations that weaken etoposide binding eliminate this resistance phenotype. We argue that the N-terminal ATP-binding pocket competes with the active site of the holoenzyme for binding etoposide both in cis and in trans with different outcomes, suggesting that each topoisomerase II monomer has two non-equivalent drug-binding sites.     

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.