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.     

Altered catalytic activity of and DNA cleavage by DNA topoisomerase II from human leukemic cells selected for resistance to VM-26

The simultaneous development of resistance to the cytotoxic effects of several classes of natural product anticancer drugs, after exposure to only one of these agents, is referred to as multiple drug resistance (MDR). At least two distinct mechanisms for MDR have been postulated: that associated with P-glycoprotein and that thought to be due to an alteration in DNA topoisomerase II activity (at-MDR). We describe studies with two sublines of human leukemic CCRF-CEM cells approximately 50-fold resistant (CEM/VM-1) and approximately 140-fold resistant (CEM/VM-1-5) to VM-26, a drug known to interfere with DNA topoisomerase II activity. Each of these lines is cross-resistant to other drugs known to affect topoisomerase II but not cross-resistant to vinblastine, an inhibitor of mitotic spindle formation. We found little difference in the amount of immunoreactive DNA topoisomerase II in 1.0 M NaCl nuclear extracts of the two resistant and parental cell lines. However, topoisomerase II in nuclear extracts of the resistant sublines is altered in both catalytic activity (unknotting) of and DNA cleavage by this enzyme. Also, the rate at which catenation occurs is 20-30-fold slower with the CEM/VM-1-5 preparations. The effect of VM-26 on both strand passing and DNA cleavage is inversely related to the degree of primary resistance of each cell line. Our data support the hypothesis that at-MDR is due to an alteration in topoisomerase II or in a factor modulating its activity.     

Topoisomerase II cleavage in chromatin

We have examined the effect of the anti-tumor drug VM-26 on purified Drosophila topoisomerase II, and used this drug to map (putative) topoisomerase II cleavage sites in chromatin. These studies indicate that VM-26 interferes with the strand breakage-rejoining catalytic cycle. VM-26 appears to stabilize the topoisomerase-II-cleavable complex and markedly enhances the formation of double-strand breaks in naked DNA. VM-26 also stimulates the formation of double-strand breaks in isolated Drosophila nuclei. Analysis of the parameters of the VM-26-stimulated cleavage reaction in nuclei strongly suggests that the double-strand scissions are generated by endogenous topoisomerase II. Finally, we have examined the distribution of (putative) cleavage sites for endogenous topoisomerase II in the chromatin of the 87A7 heat shock locus and the histone repeat unit. We have found that there are prominent VM-26-induced cleavage products from the 5' ends of the 87A7, the two heat shock protein 70 genes, and in the intergenic spacer separating these genes. Moreover, the pattern of VM-26-induced cleavage products is altered in nuclei prepared from heat-shocked cells. In the case of the histone repeat unit, only minor VM-26-induced cleavage products are observed in nuclei (in spite of the fact that experiments on naked DNA indicate that the histone repeat contains many major cleavage sites for purified topoisomerase II). These findings suggest that the nucleoprotein organization of different DNA segments may be important in determining whether specific sites are accessible to endogenous topoisomerase II in nuclei.     

Sensitivity of human type II topoisomerases to DNA damage: stimulation of enzyme-mediated DNA cleavage by abasic, oxidized and alkylated lesions

Type II topoisomerases are essential enzymes that are also the primary cellular targets for a number of important anticancer drugs. These drugs act by increasing levels of topoisomerase II-mediated DNA cleavage. Recent studies indicate that endogenous forms of DNA damage, such as abasic sites and base mismatches, also stimulate the DNA scission activity of the enzyme. To extend our understanding of how type II topoisomerases react to DNA damage, the effects of abasic sites, and oxidized and alkylated bases on DNA cleavage mediated by human topo-isomerase IIα and β were determined. Based on experiments that incorporated random abasic sites into plasmid DNA, human type II enzymes can locate lesions even within a background of several thousand undamaged base pairs. As determined by experiments that utilized site-specific forms of DNA lesions, oxidized or monoalkylated purines that allow base pairing and induce little distortion in the double helix have modest effects on topoisomerase II-mediated DNA cleavage. In contrast, 1,N⁶-ethenoadenine, a bulky lesion that disrupts base pairing, enhanced DNA cleavage ~10-fold. 1,N⁶-Ethenoadenine is the first lesion found to rival the stimulatory effects of apurinic sites on the DNA scission activity of eukaryotic type II topoisomerases.     

Local base sequence preferences for DNA cleavage by mammalian topoisomerase II in the presence of amsacrine or teniposide.

Several classes of antitumor drugs are known to stabilize topoisomerase complexes in which the enzyme is covalently bound to a terminus of a DNA strand break. The DNA cleavage sites generally are different for each class of drugs. We have determined the DNA sequence locations of a large number of drug-stimulated cleavage sites of topoisomerase II, and find that the results provide a clue to the possible structure of the complexes and the origin of the drug-specific differences. Cleavage enhancements by VM-26 and amsacrine (m-AMSA), which are representative of different classes of topoisomerase II inhibitors, have strong dependence on bases directly at the sites of cleavage. The preferred bases were C at the 3' terminus for VM-26 and A at the 5' terminus for m-AMSA. Also, a region of dyad symmetry of 12 to 16 base pairs was detected about the enzyme cleavage positions. These results are consistent with those obtained with doxorubicin, although in the case of doxorubicin, cleavage requires the presence of an A at the 3' terminus of at least one the pair of breaks that constitute a double-strand cleavage (Capranico et al., Nucleic Acids Res., 1990, 18: 6611). These findings suggest that topoisomerase II inhibitors may stack with one or the other base pair flanking the enzyme cleavage sites.     

A new compound, withangulatin A, promotes type II DNA topoisomerase-mediated DNA damage

Withangulatin A, a new compound with a known chemical structure and from the antitumor Chinese herb Physalis angulata L, was found to act on topoisomerase II to induce topoisomerase II-mediated DNA damage in vitro. It has two effective dosage ranges of approximate 0.5 and 20 microM, with about one-third the activity of 20 microM VM-26.     

Chromatin structure, not DNA sequence specificity, is the primary determinant of topoisomerase II sites of action in vivo.

In the studies reported here we have used topoisomerase II as a model system for analyzing the factors that determine the sites of action for DNA-binding proteins in vivo. To localize topoisomerase II sites in vivo we used an inhibitor of the purified enzyme, the antitumor drug VM-26. This drug stabilizes an intermediate in the catalytic cycle, the cleavable complex, and substantially stimulates DNA cleavage by topoisomerase II. We show that lysis of VM-26 treated tissue culture cells with sodium dodecyl sulfate induces highly specific double-strand breaks in genomic DNA, and we present evidence indicating that these double-strand breaks are generated by topoisomerase II. Using indirect end labeling to map the cleavage products, we have examined the in vivo sites of action of topoisomerase II in the 87A7 heat shock locus, the histone repeat, and a tRNA gene cluster at 90BC. Our analysis reveals that chromatin structure, not sequence specificity, is the primary determinant in topoisomerase II site selection in vivo. We suggest that chromatin organization may provide a general mechanism for generating specificity in a wide range of DNA-protein interactions in vivo.     

Distribution of topoisomerase II cleavage sites in simian virus 40 DNA and the effects of drugs.

The distributions of DNA cleavage sites induced by topoisomerase II in the presence or absence of specific drugs were mapped in the simian virus 40 genome. The drugs studied were 5-iminodaunorubicin, amsacrine (m-AMSA), teniposide (VM-26) and 2-methyl-9-hydroxyellipticinium; each produced a distinctive pattern of enhanced cleavage. Consistently intense cleavage, both in the presence and in the absence of drugs, occurred in the nuclear matrix-associated region. Since topoisomerase II is a major constituent of the nuclear matrix, and cleavage complexes include a covalent link between topoisomerase II and DNA, the findings suggest that topoisomerase II may function to attach DNA to the nuclear matrix. Cleavage usually occurred on both DNA strands with the expected four base-pair 5' stagger, and strong sites tended to occur within A/T runs such as have been associated with binding to the nuclear scaffold. Intense cleavage was present also in the replication termination region, but was absent from the vicinity of the replication origin. Cleavage intensities were found to change with time in a manner that depended both on the site and on the drug, suggesting that topoisomerase II can move along the DNA from a kinetically preferred site to a thermodynamically preferred site.     

Topological complexes between DNA and topoisomerase II and effects of polyamines

The polyamines spermine and spermidine were found to enhance the formation of a stable noncovalent complex between mammalian topoisomerase II and DNA. This complex is not associated with DNA strand breaks and forms to a greater extent with supercoiled than with relaxed circular or with linear DNA. Polyamine-induced complex formation is associated with a stimulation of the enzymatic relaxation of DNA supercoils. In these respects, the polyamine-enhanced complex differs from the covalent cleavable complexes stabilized by DNA intercalators such as amsacrine (m-AMSA) or epipodophylotoxins such as teniposide (VM-26). In the polyamine-enhanced complex, the topoisomerase II may be a donutlike structure topologically bound to the DNA and able to migrate and dissociate from the ends of linear DNA molecules. At relatively high concentrations, spermine (1 mM) enhances topoisomerase II induced cleavage at certain sites on the SV40 genome that could have regulatory significance.     

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.     

Position-specific effect of ribonucleotides on the cleavage activity of human topoisomerase II

Beyond the normal DNA transactions mediated by topoisomerase II, we have recently demonstrated that the cleavage activity of the two human topoisomerase II isoforms is several-fold stimulated if a ribonucleotide rather than a deoxyribonucleotide is present at the scissile phosphodiester in one strand of the substrate. Here we show that ribonucleotides exert a position-specific effect on topoisomerase II-mediated cleavage without altering the sequence specificity of the enzyme. Ribonucleotides located within the 4 bp cleavage stagger stimulate topoisomerase II-mediated cleavage, whereas ribonucleotides located outside the stagger in general have an inhibitory effect. Results obtained from competition experiments indicate that the position-specific effect of ribonucleotides on topoisomerase II activity is caused by altered substrate interaction. When cleavage is performed with substrates containing one ribonucleotide in both strands or several ribonucleotides in one strand the effect of the individual ribonucleotides on cleavage is not additive. Finally, although topoisomerase II recognizes substrates with longer stretches of ribonucleotides, an RNA/DNA hybrid where one strand is composed entirely of RNA is not cleaved by the enzyme. The positional effect of ribonucleotides on topoisomerase II-mediated cleavage shares many similarities to the positional effect exerted by either abasic sites or base mismatches, demonstrating a general influence of DNA imperfections on topoisomerase II activity.     

SUMO-1 conjugation to human DNA topoisomerase II isozymes

Topoisomerase I-mediated DNA damage induced by camptothecin has been shown to induce rapid small ubiquitin-related modifier (SUMO)-1 conjugation to topoisomerase I. In the current study, we show that topoisomerase II-mediated DNA damage induced by teniposide (VM-26) results in the formation of high molecular weight conjugates of both topoisomerase IIalpha and IIbeta isozymes in HeLa cells. Immunological characterization of these conjugates suggests that both topoisomerase IIalpha and IIbeta isozymes are conjugated to SUMO-1. The involvement of SUMO-1/UBC9 in the modification of topoisomerase II isozymes is also supported by the demonstration of physical interaction between topoisomerase II and SUMO-1/UBC9. Surprisingly, ICRF-193, which does not induce topoisomerase II-mediated DNA damage but traps topoisomerase II into a circular clamp conformation, is also shown to induce similar SUMO-1 conjugation to topoisomerase II isozymes. In addition, we show that both oxidative and heat shock stresses, which can cause protein damage, rapidly increase nuclear SUMO-1 conjugates. These studies raise the question on whether SUMO-1 conjugation to topoisomerases is an indirect result of a DNA damage response or a direct result because of protein conformational changes.     

Drug Stimulated DNA Cleavage Mediated by Cauliflower Topoisomerase II

We have investigated cauliflower (Brassica oleracea) topoisomerase II with respect to its interaction with DNA and demonstrate that the enzyme shares the characteristics of topoisomerase II purified from a variety of phylogenetically remote organisms. In the presence of the 2-nitroimidazole Ro 15-0216, cauliflower topoisomerase II-mediated DNA cleavage is extensively stimulated (approximately 20-fold) only at a site recognized as a major cleavage site for the enzyme in the absence of drug. The conservation of the enzyme's DNA specificity in the presence of Ro 15-0216 is in contrast to the effect exerted by traditional topoisomerase II inhibitors, which cause enzyme-mediated cleavage to take place at a multiple number of DNA sites. Ro 15-0216 may therefore prove useful as a tool in the elucidation of the enzyme's DNA interaction sites and its involvement in nucleic acid metabolism in plant cells.     

Minimal DNA requirement for topoisomerase II-mediated cleavage in vitro

The minimal DNA requirement for topoisomerase II-mediated DNA cleavage in vitro was determined by analyzing the interaction of the enzyme with sets of DNA substrates varying successively by single bases at the 5'- or 3'-end of either strand. A 16-base pair double-stranded region was established as the minimal duplex region required for topoisomerase II cleavage activity. The region was located symmetrically around the 4-base staggered cleavage site. Topoisomerase II-mediated cleavage within the 16-base pair core duplex, however, required single-stranded regions flanking the duplex to either the 5'- or 3'-sides, or an extension at both ends of the duplex with 1 or more base pairs.     

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.     

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.     

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)