DNA minor groove binding agents interfere with topoisomerase II mediated lesions induced by epipodophyllotoxin derivative VM-26 and acridine derivative m-AMSA in nuclei from L1210 cells

This study demonstrated that agents capable of interacting with the minor groove in nuclear DNA interfere with topoisomerase II mediated effects of antitumor drugs such as VM-26 and m-AMSA. Distamycin, Hoechst 33258, and DAPI were used as agents capable of AT-specific binding in the minor groove of DNA while producing no profound long-range distortion of DNA structure. In intact nuclei from L1210 cells, these minor groove binders inhibited the induction of topoisomerase II mediated DNA damage (DNA-protein cross-links and DNA double-strand breaks) by VM-26 and m-AMSA. The inhibitory effects of distamycin reflected prevention of formation of new lesions but not reversal of preexisting damage. The minor groove binders did not differentiate between lesions induced by an intercalator, m-AMSA, or by a DNA-nonbinding drug, VM-26. All three groove binders inhibited DNA breaks more strongly than DNA-protein cross-links. The inhibitory potency correlated with the size of minor groove binders and the size of their DNA-binding sites: distamycin (5 bp) greater than Hoechst 33258 (4 bp) greater than DAPI (3 bp). The results showed that DNA minor groove binders are a new type of modulators of the action of topoisomerase II targeted drugs.     

DNA binding properties of minor groove binders and their influence on the topoisomerase II cleavage reaction

We present titrations of the human δβ-globin gene region with DNA minor groove binders netropsin, bisnetropsin, distamycin, chromomycin and four bis-quaternary ammonium compounds in the presence of calf thymus topoisomerase II and DNase I. With increasing ligand concentration, stimulation and inhibition of enzyme activity were detected and quantitatively evaluated. Additionally we show a second type of stimulation, the appearance of strong new topoisomerase II cleavage sites at high ligand concentrations. The specific binding sites of the minor groove binders of the DNA sequence and their microscopic binding constants were determined from DNase I footprints. A binding mechanism for minor groove binders is proposed in order to explain these results especially when ligand concentration is increased. © 1998 John Wiley & Sons, Ltd.     

Effects of the DNA intercalators 4'-(9-acridinylamino)methanesulfon-m-anisidide and 2-methyl-9-hydroxyellipticinium on topoisomerase II mediated DNA strand cleavage and strand passage

DNA topoisomerase II is believed to be the enzyme that produces the protein-associated DNA strand breaks observed in mammalian cell nuclei treated with various intercalating agents. Two intercalators--4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA, amsacrine) and 2-methyl-9-hydroxyellipticinium (2-Me-9-OH-E+)--differ in their effects on protein-associated double-strand breaks in isolated nuclei. m-AMSA stimulates their production at all concentrations, whereas 2-Me-9-OH-E+ stimulates at low concentrations and inhibits at high concentrations. We have reproduced these differential effects in experiments carried out in vitro with purified L1210 DNA topoisomerase II, and we have found that concentrations of 2-Me-9-OH-E+ above 5 microM prevent the trapping of DNA-topoisomerase II cleavable complexes irrespective of the presence of m-AMSA. It also stimulated topoisomerase II mediated DNA strand passage, again with or without inhibitory amounts of m-AMSA (this result suggests that extensive intercalation by 2-Me-9-OH-E+ destabilized the cleavable complexes). From these data, it is concluded that intercalator-induced protein-associated DNA strand breaks observed in intact eukaryotic cells and isolated nuclei are generated by DNA topoisomerase II and that intercalators can affect mammalian DNA topoisomerase II in more than one way. They can trap cleavable complexes and inhibit DNA topoisomerase II mediated DNA relaxation (m-AMSA and low concentrations of 2-Me-9-OH-E+) or destabilize cleavable complexes and stimulate DNA relaxation (high concentrations of 2-Me-9-OH-E+).     

Design,synthesis and biological evaluation of a novel series of anthrapyrazoles linked with netropsin-like oligopyrrole carboxamides as anticancer agents

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. Combilexin molecules, which combine DNA minor groove binding and intercalating functionalities, have the potential for increased DNA binding affinity and increased selectivity due to their dual mode of DNA binding. This study describes the synthesis of DNA minor groove binder netropsin analogs containing either one or two N-methylpyrrole carboxamide groups linked to DNA-intercalating anthrapyrazoles. Those hybrid molecules which had both two N-methylpyrrole groups and terminal (dimethylamino)alkyl side chains displayed submicromolar cytotoxicity towards K562 human leukemia cells. The combilexins were also evaluated for DNA binding by measuring the increase in DNA melting temperature, for DNA topoisomerase IIα-mediated double strand cleavage of DNA, for inhibition of DNA topoisomerase IIα decatenation activity, and for inhibition of DNA topoisomerase I relaxation of DNA. Several of the compounds stabilized the DNA–topoisomerase IIα covalent complex indicating that they acted as topoisomerase IIα poisons. Some of the combilexins had higher affinity for DNA than their parent anthrapyrazoles. In conclusion, a novel group of compounds combining DNA intercalating anthrapyrazole groups and minor groove binding netropsin analogs have been designed, synthesized and biologically evaluated as possible novel anticancer agents.     

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.     

Oligonucleotide-minor groove binder conjugates and their complexes with complementary DNA: effect of conjugate structural factors on the thermal stability of duplexes

Synthetic polycarboxamide minor groove binders (MGB) consisting of N-methylpyrrole (Py), N-methylimidazole (Im), N-methyl-3-hydroxypyrrole (Hp) and beta-alanine (beta) show strong and sequence-specific interaction with the DNA minor groove in side-by-side antiparallel or parallel orientation. Two MGB moieties covalently linked to the same terminal phosphate of one DNA strand stabilize DNA duplexes formed by this strand with a complementary one in a sequence-specific manner, similarly to the corresponding mono-conjugated hairpin structures. The series of conjugates with the general formula Oligo-(L-MGB-R)m was synthesized, where m = 1 or 2, L = linker, R = terminal charged or neutral group, MGB = -(Py)n-, -(Im)n- or -[(Py/Im)n-(CH2)3CONH-(Py/Im)n-] and I < n < 5. Using thermal denaturation, we studied effects of structural factors such as m and n, linker L length, nature and orientation of the MGB monomers, the group R and the backbone (DNA or RNA), etc. on the stability of the duplexes. Structural factors are more important for linear and hairpin monophosphoroamidates than for parallel bis-phosphoroamidates. No more than two oligocarboxamide strands can be inserted into the duplex minor groove. Attachment of the second sequence-specific parallel ligand [-L(Py)4R] to monophosphoroamidate conjugate CGTTTATT-L(Py)4R leads to the increase of the duplex Tm, whereas attachment of [-L(Im)4R] leads to its decrease. The mode of interaction between oligonucleotide duplex and attached ligands could be different (stacking with the terminal A:T pair of the duplex or its insertion into the minor groove) depending on the length and structure of the MGB.     

Sequence-specific recognition of double-stranded DNA by synthetic minor groove binder conjugates. toward the construction of artificial site-specific deoxyribonucleases

Bis-conjugates of hairpin N-methylpyrrole/N-methylimidazole oligocarboxamide minor groove binders (MGB) possessing enhanced affinity and sequence-specificity for dsDNA were synthesized. Two hairpin MGBs were connected by their N-termini via an aminodiacetate linker. The binding of bis-MGB conjugates to the target DNA was studied by gel mobility retardation, footprinting, and circular dichroism; their affinity and binding mode in the DNA minor groove were determined. In order to functionalize the bis-MGB conjugates, DNA-cleaving agents such as phenanthroline or bipyridine were attached. Effective site-specific cleavage of target DNA in the presence of Cu(2+) ions was observed.     

DNA abasic lesions in a different light: solution structure of an endogenous topoisomerase II poison

Topoisomerase II is the target for several anticancer drugs that "poison" the enzyme and convert it to a cellular toxin by increasing topoisomerase II-mediated DNA cleavage. In addition to these "exogenous topoisomerase II poisons," DNA lesions such as abasic sites act as "endogenous poisons" of the enzyme. Drugs and lesions are believed to stimulate DNA scission by altering the structure of the double helix within the cleavage site of the enzyme. However, the structural alterations that enhance cleavage are unknown. Since abasic sites are an intrinsic part of the genetic material, they represent an attractive model to assess DNA distortions that lead to altered topoisomerase II function. Therefore, the structure of a double-stranded dodecamer containing a tetrahydrofuran apurinic lesion at the +2 position of a topoisomerase II DNA cleavage site was determined by NMR spectroscopy. Three major features distinguished the apurinic structure ( = 0.095) from that of wild-type ( = 0.077). First, loss of base stacking at the lesion collapsed the major groove and reduced the distance between the two scissile phosphodiester bonds. Second, the apurinic lesion induced a bend that was centered about the topoisomerase II cleavage site. Third, the base immediately opposite the lesion was extrahelical and relocated to the minor groove. All of these structural alterations have the potential to influence interactions between topoisomerase II and its DNA substrate.     

In vivo topoisomerase II cleavage of the Drosophila histone and satellite III repeats: DNA sequence and structural characteristics.

We have identified two classes of in vivo topoisomerase II cleavage sites in the Drosophila histone gene repeat. One class co-localizes with DNase I-hypersensitive regions and another novel class maps to a subset of consecutive nucleosome linker sites in the scaffold-associated region (SAR) of the histone gene loop. Prominent topoisomerase II cleavage is also observed in one of the linker regions of the two nucleosomes spanning satellite III, a centromeric SAR-like DNA sequence with a repeat length of 359 bp. At the sequence level, in vivo topoisomerase II cleavage is highly site specific. Comparison of 10 nucleosome linker sites defines an in vivo cleavage sequence whose major characteristic is a prominent GC-rich core. These GC-rich cleavage sites are flanked by extensive arrays of oligo(dA).oligo(dT) tracts characteristic of SAR sequences. Treatment of cells with distamycin selectively enhances cleavage at nucleosome linker sites of the SAR and satellite regions, suggesting that AT-rich sequences flanking cleavage sites may be involved in determining topoisomerase II activity in the cell. These observations provide evidence for the association of topoisomerase II with SARS in vivo.     

Site-specific DNA cleavage by Chlorella virus topoisomerase II

The DNA cleavage reaction of topoisomerase II is central to the catalytic activity of the enzyme and is the target for a number of important anticancer drugs. Unfortunately, efforts to characterize this fundamental reaction have been limited by the low levels of DNA breaks normally generated by the enzyme. Recently, however, a type II topoisomerase with an extraordinarily high intrinsic DNA cleavage activity was isolated from Chlorella virus PBCV-1. To further our understanding of this enzyme, the present study characterized the site-specific DNA cleavage reaction of PBCV-1 topoisomerase II. Results indicate that the viral enzyme cleaves DNA at a limited number of sites. The DNA cleavage site utilization of PBCV-1 topoisomerase II is remarkably similar to that of human topoisomerase IIalpha, but the viral enzyme cleaves these sites to a far greater extent. Finally, PBCV-1 topoisomerase II displays a modest sensitivity to anticancer drugs and DNA damage in a site-specific manner. These findings suggest that PBCV-1 topoisomerase II represents a unique model with which to dissect the DNA cleavage reaction of eukaryotic type II topoisomerases.     

Amiloride intercalates into DNA and inhibits DNA topoisomerase II

Amiloride is capable of inhibiting DNA synthesis in mammalian cells in culture. Recent evidence indicates that the enzyme, DNA topoisomerase II, is probably required for DNA synthesis to occur in situ. In experiments to determine the mechanism of inhibition of DNA synthesis by amiloride, we observed that amiloride inhibited both the catalytic activity of purified DNA topoisomerase II in vitro and DNA topoisomerase II-dependent cell functions in vivo. Many compounds capable of inhibiting DNA topoisomerase II are DNA intercalators. Thus, we performed studies to determine if and how amiloride bound to DNA. Results indicated that amiloride 1) shifted the thermal denaturation profile of DNA, 2) increased the viscosity of linear DNA, and 3) unwound circular DNA, all behavior consistent with a DNA intercalation mechanism. Furthermore, quantitative and qualitative measurements of amiloride fluorescence indicated that amiloride (a) bound reversibly to purified DNA under conditions of physiologic ionic strength, and (b) bound to purified nuclei in a highly cooperative manner. Lastly, amiloride did not promote the cleavage of DNA in the presence of DNA topoisomerase II, indicating that the mechanism by which amiloride inhibited DNA topoisomerase II was not through the stabilization of a "cleavable complex" formed between DNA topoisomerase II, DNA, and amiloride. The ability of amiloride to intercalate with DNA and inhibit topoisomerase II is consistent with the proposed planar, hydrogen-bonded, tricyclic nature of amiloride's most stable conformation. Thus, DNA and DNA topoisomerase II must be considered as new cellular targets of amiloride action.     

A functional linker in human topoisomerase I is required for maximum sensitivity to camptothecin in a DNA relaxation assay.

Human topoisomerase I is composed of four major domains: the highly charged NH(2)-terminal region, the conserved core domain, the positively charged linker domain, and the highly conserved COOH-terminal domain. Near complete enzyme activity can be reconstituted by combining recombinant polypeptides that approximate the core and COOH-terminal domains, although DNA binding is reduced somewhat for the reconstituted enzyme (Stewart, L., Ireton, G. C., and Champoux, J. J. (1997) J. Mol. Biol. 269, 355-372). A reconstituted enzyme comprising the core domain plus a COOH-terminal fragment containing the complete linker region exhibits the same biochemical properties as a reconstituted enzyme lacking the linker altogether, and thus detachment of the linker from the core domain renders the linker non-functional. The rate of religation by the reconstituted enzyme is increased relative to the forms of the enzyme containing the linker indicating that in the intact enzyme the linker slows religation. Relaxation of plasmid DNA by full-length human topoisomerase I or a 70-kDa form of the enzyme that is missing only the non-essential NH(2)-terminal domain (topo70) is inhibited approximately 16-fold by the anticancer compound, camptothecin, whereas the reconstituted enzyme is nearly resistant to the inhibitory effects of the drug despite similar affinities for the drug by the two forms of the enzyme. Based on these results and in light of the crystal structure of human topoisomerase I, we propose that the linker plays a role in hindering supercoil relaxation during the normal relaxation reaction and that camptothecin inhibition of DNA relaxation depends on a direct effect of the drug on DNA rotation that is also dependent on the linker.     

Decreased nuclear matrix DNA topoisomerase II in human leukemia cells resistant to VM-26 and m-AMSA

CEM leukemia cells selected for resistance to VM-26 (CEM/VM-1) are cross-resistant to various other DNA topoisomerase II inhibitors but not to Vinca alkaloids. Since DNA topoisomerase II is a major protein of the nuclear matrix, we asked if alterations in nuclear matrix topoisomerase II might be important in this form of multidrug resistance. Pretreatment of drug-sensitive CEM cells for 2 h with either 5 microM VM-26 or 3 microM m-AMSA reduced the specific activity of newly replicated DNA on the nuclear matrix by 75 and 50%, respectively, relative to that of the bulk DNA. However, neither VM-26 nor m-AMSA affected the relative specific activity of nascent DNA isolated from the nuclear matrices of drug-resistant CEM/VM-1 cells. The decatenating and unknotting activities of DNA topoisomerase II were 6- and 7-fold lower, respectively, in the nuclear matrix preparations from the CEM/VM-1 cells compared to parental CEM cells. Western blot analysis revealed that the amount of immunoreactive topoisomerase II in the nuclear matrices of the CEM/VM-1 cells was decreased 3.2-fold relative to that in CEM cells, but there was no significant difference in the amount of enzyme present in the nonmatrix (1.5 M salt soluble) fractions of nuclei from these cell lines. Increasing the NaCl concentration used in the matrix isolation procedure from 0.2 to 1.8 M resulted in a progressive decrease in the specific activity of topoisomerase II in matrices of CEM/VM-1 but not CEM cells, which suggested that the association of the enzyme with the matrix is altered in the resistant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of intercalator-dependent protein-linked DNA strand cleavage activity from cell nuclei and identification as topoisomerase II

DNA intercalating agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) have previously been found to induce in mammalian cells the formation of protein-associated DNA single- and double-strand breaks. In the current work, an activity characterized by the production of DNA-protein links associated with DNA strand breaks and by stimulation by m-AMSA was isolated from L1210 cell nuclei and was shown to be due to topoisomerase II. Nuclei were extracted with 0.35 M NaCl, and the extract was fractionated by gel filtration, DNA-cellulose chromatography, and glycerol gradient centrifugation. A rapid filter binding assay was devised to monitor the fractionation procedure on the basis of DNA-protein linking activity. The active DNA-cellulose fraction contained both topoisomerase I and topoisomerase II whereas the glycerol gradient purified material contained only topoisomerase II activity. The properties of the active material were studied at both stages of purification. m-AMSA enhanced the formation of complexes between purified topoisomerase II and SV40 DNA in which the DNA sustained a single- or double-strand cut and the enzyme was covalently linked to the 5' terminus of the DNA. This action was further enhanced by ATP, as well as by nonhydrolyzable ATP analogues. m-AMSA inhibited the topoisomerization and catenation reactions of topoisomerase II, probably because of trapping of the enzyme-DNA complexes. The activity showed a dependence on the type of DNA intercalators used, analogous to what was previously observed in intact cells. m-AMSA had no effect on topoisomerase I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Single mutation in the linker domain confers protein flexibility and camptothecin resistance to human topoisomerase I

DNA topoisomerase I relaxes supercoiled DNA by the formation of a covalent intermediate in which the active-site tyrosine is transiently bound to the cleaved DNA strand. The antineoplastic agent camptothecin specifically targets DNA topoisomerase I, and several mutations have been isolated that render the enzyme camptothecin-resistant. The catalytic and structural dynamical properties of a human DNA topoisomerase I mutant in which Ala-653 in the linker domain was mutated into Pro have been investigated. The mutant is resistant to camptothecin and in the absence of the drug displays a cleavage-religation equilibrium strongly shifted toward religation. The shift is mainly because of an increase in the religation rate relative to the wild type enzyme, indicating that the unperturbed linker is involved in slowing religation. Molecular dynamics simulation indicates that the Ala to Pro mutation increases the linker flexibility allowing it to sample a wider conformational space. The increase in religation rate of the mutant, explained by means of the enhanced linker flexibility, provides an explanation for the mutant camptothecin resistance.     

A truncated form of DNA topoisomerase IIbeta associates with the mtDNA genome in mammalian mitochondria.

Despite the likely requirement for a DNA topoisomerase II activity during synthesis of mitochondrial DNA in mammals, this activity has been very difficult to identify convincingly. The only DNA topoisomerase II activity conclusively demonstrated to be mitochondrial in origin is that of a type II activity found associated with the mitochondrial, kinetoplast DNA network in trypanosomatid protozoa [Melendy, T., Sheline, C., and Ray, D.S. (1988) Cell 55, 1083-1088; Shapiro, T.A., Klein, V.A., and Englund, P.A. (1989) J. Biol. Chem.264, 4173-4178]. In the present study, we report the discovery of a type DNA topoisomerase II activity in bovine mitochondria. Identified among mtDNA replicative proteins recovered from complexes of mtDNA and protein, the DNA topoisomerase relaxes a negatively, supercoiled DNA template in vitro, in a reaction that requires Mg2+ and ATP. The relaxation activity is inhibited by etoposide and other inhibitors of eucaryotic type II enzymes. The DNA topoisomerase II copurifies with mitochondria and directly associates with mtDNA, as indicated by sensitivity of some mtDNA circles in the isolated complex of mtDNA and protein to cleavage by etoposide. The purified activity can be assigned to a approximately 150-kDa protein, which is recognized by a polyclonal antibody made against the trypanosomal mitochondrial topo II enzyme. Mass spectrometry performed on peptides prepared from the approximately 150-kDa protein demonstrate that this bovine mitochondrial activity is a truncated version of DNA topoisomerase IIbeta, one of two DNA topoisomerase II activities known to exist in mammalian nuclei.     

Cadmium is a catalytic inhibitor of DNA topoisomerase II

Cadmium (Cd²⁺) is a highly toxic and carcinogenic metal that is an environmental and occupational hazard. DNA topoisomerase II is an essential nuclear enzyme and its inhibition can result in the formation of genotoxic and recombinogenic DNA double strand breaks. In this study we showed that cadmium chloride strongly inhibited the DNA decatenation activity of human topoisomerase IIα in the low micromolar concentration range and that its inhibitory effects were reduced by glutathione. Because the activity of topoisomerase II is strongly inhibited by thiol-reactive compounds this result suggested that cadmium may be binding to critical topoisomerase II cysteine thiols. Cadmium, however, did not stabilize DNA-topoisomerase II covalent complexes, as measured by the lack of formation of DNA double strand breaks. Hence, it is not likely to be a topoisomerase II poison. Consistent with the idea that cadmium cytotoxicity may be modulated by glutathione levels, buthionine sulfoximine pretreatment to decrease glutathione levels resulted in a greatly increased cadmium-induced cytotoxicity in K562 cells. The results of this study suggest that cadmium may exert some of its cell growth inhibitory, and possibly its toxicity and carcinogenicity, by inhibiting topoisomerase IIα through reaction with critical cysteine thiols.     

Human autoantibody to topoisomerase II

The rheumatic diseases are characterized by the production of autoantibodies that are usually directed against components of the cell nucleus. In this communication, we describe autoantibodies that recognize DNA topoisomerase II (anti-topoII) present in the serum of a patient with systemic lupus erythematosus. Several lines of evidence indicate that this antibody recognizes topoisomerase II. First, it binds to the native enzyme in soluble extracts prepared from isolated chromosomes and effectively depletes such extracts of active enzyme. Second, the serum binds to topoisomerase II in immunoblots of mitotic chromosomes and chromosome scaffolds. Finally, the antiserum binds strongly to a fusion protein encoded by a cloned cDNA and expressed in Escherichia coli that (based on immunological evidence) represents the carboxy-terminal portion of chicken topoisomerase II. Autoantibodies such as the one described here may provide useful reagents for the study of human topoisomerase II.