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Telomeres of human chromosomes contain a G-rich 3'-overhang that adopts an intramolecular G-quadruplex structure in vitro which blocks the catalytic reaction of telomerase. Agents that stabilize G-quadruplexes have the potential to interfere with telomere replication by blocking the elongation step catalyzed by telomerase and can therefore act as antitumor agents. We have identified by Fluorescence Resonance Energy Transfer a new series of quinoline-based G-quadruplex ligands that also exhibit potent and specific anti-telomerase activity with IC50 in the nanomolar concentration range. Long term treatment of tumor cells at subapoptotic dosage induces a delayed growth arrest that depends on the initial telomere length. This growth arrest is associated with telomere erosion and the appearance of the senescent cell phenotype (large size and expression of beta-galactosidase activity). Our data show that a G-quadruplex interacting agent is able to impair telomerase function in a tumor cell thus providing a basis for the development of new anticancer agents.

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt an intramolecular G-quadruplex structure in vitro, which has been shown to inhibit telomerase activity. The C-rich sequence can also adopt a quadruplex (intercalated) structure (i-DNA). Two acridine derivatives were shown to increase the melting temperature of the G- quadruplex and the C-quadruplex at 1 microM dye concentration. The increase in Tm value of the G-quadruplex was associated with telomerase inhibition in vitro. The most active compound, "BisA", showed an IC(50) value of 0.75 microM in a standard TRAP assay.

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5' and 3' ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2-20 degrees C at 1 microM dye concentration. This increase in T(m) value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC(50) value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we describe ethidium derivatives that stabilize G-quadruplexes. These molecules were shown to increase the melting temperature of an intramolecular quadruplex structure, as shown by fluorescence and absorbance measurements, and to facilitate the formation of intermolecular quadruplex structures. In addition, these molecules may be used to reveal the formation of multi-stranded DNA structures by standard fluorescence imaging, and therefore become fluorescent probes of quadruplex structures. This recognition was associated with telomerase inhibition in vitro: these derivatives showed a potent anti-telomerase activity, with IC(50) values of 18-100 nM in a standard TRAP assay.

Because of their role in the control of the topological state of DNA, topoisomerases are ubiquitous and vital enzymes, which participate in nearly all events related to DNA metabolism including replication and transcription. We show here that human topoisomerase I (Topo I) plays an unexpected role of 'molecular matchmaker' for G-quartet formation. G-quadruplexes are multi-stranded structures held together by square planes of four guanines ('G-quartets') interacting by forming Hoogsteen hydrogen bonds. Topo I is able to promote the formation of four-stranded intermolecular DNA structures when added to single-stranded DNA containing a stretch of at least five guanines. We provide evidence that these complexes are parallel G-quartet structures, mediated by tetrads of hydrogen-bonded guanine. In addition, Topo I binds specifically to pre-formed parallel and anti-parallel G4-DNA.

Human topoisomerase IIIalpha (hTopo IIIalpha), the recently identified first member of the topoisomerase IA subfamily in humans, has a central domain which is highly homologous to the yeast topoisomerase III, but an overall organization closer to that of Escherichia coli DNA topoisomerase I. In order to determine the properties of hTopo IIIalpha, compared to those of other topoisomerase IA subfamily members, we purified this enzyme to near homogeneity, together with an active site-mutant Y337F. We show that hTopo IIIalpha is able to relax negatively supercoiled DNA in a distributive manner, leading to the total disappearance of the initial substrate and the appearance of intermediate topoisomers. This DNA relaxation activity is magnesium-dependent, although a low concentration of MgCl2is sufficient to obtain efficient catalysis. 32P-transfer experiments demonstrated that hTopo IIIalpha is able to cleave a single-stranded oligonucleotide and to bind covalently to the 5'-end of the cleaved DNA. Addition of 0.5 M NaCl reverses the reaction, leading to the religation of the oligo-nucleotide. Experiments utilizing several different single-stranded oligonucleotides permitted us to map several cleavage sites and to deduce a consensus sequence for DNA cleavage (CANNN downward arrow), which is different from that for other members of the Topo IA subfamily.

Many antitumor agents contain a carbohydrate side chain appended to a DNA-intercalating chromophore. This is the case with anthracyclines such as daunomycin and also with indolocarbazoles including the antibiotic rebeccamycin and its tumor active analog, NB506. In each case, the glycoside residue plays a significant role in the interaction of the drug with the DNA double helix. In this study we show that the DNA-binding affinity and sequence selectivity of a rebeccamycin derivative can be enhanced by replacing the glucose residue with a 2'-aminoglucose moiety. The drug-DNA interactions were studied by thermal denaturation, fluorescence, and footprinting experiments. The thermodynamic parameters indicate that the newly introduced amino group on the glycoside residue significantly enhanced binding to DNA by increasing the contribution of the polyelectrolyte effect to the binding free energy, but does not appear to participate in any specific molecular contacts. The energetic contribution of the amino group of the rebeccamycin analog was found to be weaker than that of the sugar amino group of daunomycin, possibly because the indolocarbazole derivative is only partially charged at neutral pH. Topoisomerase I-mediated DNA cleavage studies reveal that the OH-->NH2 substitution does not affect the capacity of the drug to stabilize enzyme-DNA covalent complexes. Cytotoxicity studies with P388 leukemia cells sensitive or resistant to camptothecin suggest that topoisomerase I represents a privileged intracellular target for the studied compounds. The role of the sugar amino group is discussed. The study provides useful guidelines for the development of a new generation of indolocarbazole-based antitumor agents.

Two anthraquinone derivatives of the anticancer drugs mitoxantrone and ametantrone were examined for their ability to bind to DNA and to modulate the formation of topoisomerase-DNA cleavable complexes in vitro. The guanidinium groups introduced at the termini of the two aminoethylamino side chains of mitoxantrone can reinforce the interaction with DNA as judged from thermal denaturation studies with calf thymus DNA and polynucleotides. Footprinting experiments indicate that the binding to DNA of compound SR107 lacking the 5,8-hydroxyl substituents is essentially nonspecific whereas its congener SR 103 interacts preferentially with GC-rich sequences, particularly those containing 5'-(A/T)CG sites. Compound SR103, which bears two hydroxyl groups on the anthraquinone chromophore, promotes the cleavage of DNA by topoisomerase II and is cytotoxic toward human KB carcinoma cells in vitro. In contrast, the analogue SR107, which lacks OH groups, has no effect on topoisomerase II and is not cytotoxic.

DNA topoisomerase I has been shown to be an important therapeutic target in cancer chemotherapy for the camptothecins as well as for indolocarbazole antibiotics such as BE-13793C and its synthetic derivatives NB-506 and ED-110 [Yoshinari et al. (1993) Cancer Res. 53, 490-494]. To investigate the mechanism of topoisomerase I inhibition by indolocarbazoles, we have studied the induction of DNA cleavage by purified mammalian topoisomerase I mediated by the antitumor antibiotic rebeccamycin and a series of 20 indolocarbazole derivatives. The compounds tested bear (i) various functional groups on the non-indolic moiety (X = CO, CH2, CHOH), (ii) a hydrogen or a chlorine atom at positions 1 and 11 (R2), and (iii) different substituents on the maleimido function (R1 = H, OH, NH2, NHCHO). Half of the ligands have the same carbohydrate moiety as rebeccamycin whereas the other ligands have no sugar residue. The inhibitory potency of the test compounds was assessed in vitro by comparing the cleavage of [32P]-labeled restriction fragments by the enzyme in the absence and presence of the drug. In addition, the DNA-binding properties of these compounds were investigated by means of complementary spectroscopic techniques including electric linear dichroism, and the DNA sequence selectivity was probed by DNase I footprinting. The study shows that the sugar residue attached to the indolocarbazole chromophore is critical for the drug ability to interfere with topoisomerase I as well as for the formation of intercalation complexes. Structure-activity relationships indicate that the presence of chlorine atoms significantly reduces the effects on topoisomerase I whereas the substituents on the maleimido function and the functional group on the non-indolic moiety can be varied without reduction of activity. The results suggest that the inhibition of topoisomerase I by indolocarbazoles arises in part from their ability to interact with DNA. Analysis of the base preferences around topoisomerase I cleavage sites in various restriction fragments indicated that, in a manner similar to camptothecin, the rebeccamycin analogue R-3 stabilized topoisomerase I preferentially at sites having a T and a G on the 5' and 3' sides of the cleaved bond, respectively. By analogy with models previously proposed for camptothecin and numerous topoisomerase II inhibitors which intercalate into DNA, a stacking model for the interaction between DNA, topoisomerase I and indolocarbazoles is proposed. These findings provide guidance for the development of new topoisomerase I-targeted antitumor indolocarbazole derivatives.

A conjugate molecule was synthesized by linking the DNA-intercalative antitumor drug 4'-(9-acridinylamino)methanesulfon-manisidide (mAMSA) via a 4-carboxamide side chain to a dipyrrolecarboxamide moiety structurally related to the minor groove-binding antibiotic netropsin. The molecule (netropsin/ mAMSA) behaves as a threading intercalator. Its netropsin-like tail becomes located in the minor groove of the double helix and serves to drive the hybrid molecule preferentially to AT-rich sites on various DNA fragments as revealed by DNase I footprinting. The hybrid retains the susceptibility to copper-dependent oxidation characteristic of the parent mAMSA moiety as well as its ability to generate oxygen radicals, which can mediate DNA damage, mainly at cytidine and guanosine nucleotides. It also retains the property of stimulating the formation of cleavable complexes with DNA in the presence of topoisomerase II, but its netropsin-like moiety confers little or no influence on the reaction with topoisomerase I. Although netropsin/mAMSA is less potent than mAMSA at producing cleavable complexes with topoisomerase II, it promotes the appearance of cleavage sites at much the same nucleotide sequences as does the parent compound. The dipyrrolecarboxamide tail is not silent, however, since it modifies the concentration-dependence of cleavable complex formation.

Cytotoxicity of several classes of antitumor DNA intercalators is thought to result from disturbance of DNA metabolism following trapping of the nuclear enzyme DNA topoisomerase II as a covalent complex on DNA. Here, molecular interactions of the potent antitumor drug amsacrine (m-AMSA), an inhibitor of topoisomerase II, within living K562 cancer cells have been studied using surface-enhanced Raman (SER) spectroscopy. The work is based on data of the previously performed model SER experiments dealing with amsacrine/DNA, drug/topoisomerase II and drug/DNA/topoisomerase II complexes in aqueous buffer solutions. The SER data indicated two kinds of amsacrine interactions in the model complexes with topoisomerase II alone or within ternary complex: non-specific (via the acridine moiety) and specific to the enzyme conformation (via the side chain of the drug). These two types of interactions have been both revealed by the micro-SER spectra of amsacrine within living K562 cancer cells. Our data suppose the specific interactions of amsacrine with topoisomerase II via the side chain of the drug (particular feature of the drug/topoisomerase II and ternary complexes) to be crucial for its inhibitory activity.

A series of amidoethylamino-anthraquinone derivatives bearing either one or two salen (bis(salicylidene)ethylenediamine) moieties complexed with CuII or NiII have been synthesized, and their DNA-binding and cleaving properties examined. The effects of the mono- and di-substituted anthracenedione-salen conjugates on DNA cleavage mediated by topoisomerases I and II have also been determined, as well as their cytotoxicity toward human KB cells. The anthraquinone-salen. NiII conjugates bind to GC-rich sequences in DNA, but do not cleave the macromolecule. By contrast, the anthraquinone-salen. CuII hybrids do not recognize particular nucleotide sequences but efficiently induce single-strand breaks in DNA after activation. The 5,8-dihydroxy-anthraquinone conjugates are more cytotoxic and more potent toward topoisomerase II than the non-hydroxylated analogues, but they are less cytotoxic than the salen-free anthraquinones. The attachment of a salen. CuII complex to the anthraquinone chromophore can confer DNA cleaving properties in vitro, but this is at the expense of cytotoxic activity. Anthraquinone-salen. CuII complexes may find useful employ as footprinting probes for investigating ligand-DNA interactions.

A number of acridine derivatives, including the clinical antileukaemia agent amsacrine and the experimental agent DACA (N-[2-(dimethylamino)ethyl]acridine-4-carboxamide), target the enzyme topoisomerase II. We demonstrate here that DACA induces DNA cleavage in the presence of topoisomerase I as well as of topoisomerase II. We also investigate a series of acridine derivatives which link amsacrine to DACA in terms of DNA binding, topoisomerase poisoning and biological activity. The presence of an acridine 4-linked N-2-(dimethylamino)ethyl group provides both a pronounced G-C preference for DNA binding and activity towards topoisomerase I. The removal of the anilino side chain of amsacrine, in combination with the presence of the N-2-(dimethylamino)ethyl group, provides in vitro biological activity against "atypical" multidrug resistant leukaemia lines with low topoisomerase II activity. Among these compounds, suppression of the ionisation of the acridine nitrogen to produce the compound DACA is associated with experimental activity against solid tumours. The addition of an acridine 2-chloro substituent to DACA suppresses the stimulation of topoisomerase II-dependent DNA cleavage but increases stimulation of topoisomerase I cleavage. 2-Substitution also increases activity against the "atypical" multidrug resistant cell lines. Overall, the results suggest that augmentation of topoisomerase I-dependent activity in this series by appropriate chemical substitution in this series leads to circumvention of topoisomerase II-mediated multidrug resistance.

Molecular interactions of intoplicine, dual DNA-topoisomerases (Topo) I and II inhibitor, with topoisomerases, plasmid DNA, in ternary cleavable complexes with enzymes and plasmid DNA, and in the reversed cleavable complexes were examined by means of surface-enhanced Raman scattering (SERS) and CD spectroscopy and by biochemical techniques. Detailed spectral analysis of intoplicine derivatives allowed us to assign SERS vibrational modes of chromophores and to propose the models for these complexes. Intoplicine was found to be able to interact specifically with the Topo II alone, but with Topo I only when in the presence of DNA. It shows at least two modes of binding to the DNA: the first was found to be dominant for its derivative 1c (most potent Topo I inhibitor), and the second was dominant for derivative 2a (most potent Topo II inhibitor). The possibility of forming these two types of complexes simultaneously is suggested to be one of the main factors enabling the drug to be a dual Topo I and Topo II inhibitor. The "deep intercalation mode" of the drug from the DNA minor groove with the long axis of the chromophore oriented roughly parallel to the dyad axis has been suggested to be responsible for induction of distortions of the DNA structure by the intercalating drug. Being involved in the formation of Topo I-mediated cleavable ternary complex, the molecules participating in the deep intercalation mode within the DNA do not change their molecular interactions as compared with their complex with the DNA alone. The stabilization of the Topo I-mediated cleavable complex was shown to be followed by the local denaturation of DNA in the AT-rich regions of the helix. When the ternary cleavable complex was reversed, the drug was shown to be in the complex with the plasmid. The "outside binding mode" from the DNA major groove via the hydroxyl group of the A-ring of the chromophore has been suggested to be responsible for Topo II inhibition. These molecules did not induce significant distortions of the DNA structure. Being involved in the formation of Topo II-mediated cleavable ternary complex, the drug changed its molecular interactions as compared with the complex with DNA alone.(ABSTRACT TRUNCATED AT 400 WORDS)

The surface-enhanced Raman scattering spectra of the new antitumoral agent, intoplicine (RP 60475, NSC 645008), and those of its complexes with DNA and topoisomerase II in vitro and in K562 cancer cells were obtained. Intoplicine was found to unwind DNA and to inhibit purified calf thymus topoisomerase II via a stabilization of the ternary cleavable complex. The intensity of the surface-enhanced Raman scattering spectrum of intoplicine was not modified by the addition of plasmid pBR322 or calf thymus DNA. In the complex of this antitumor agent with topoisomerase II, the signal of intoplicine was completely abolished, indicating that at least some portion of intoplicine binds to an internal part of the enzyme. During the formation of the ternary complex, intoplicine was released from the interior of the protein and formed hydrogen bonds via its hydroxyl and/or amino groups. Similar modifications of the intoplicine spectra were found by microsurface-enhanced Raman scattering spectroscopy of the compound in the nucleus of treated K562 cells. In contrast, intoplicine was found to be in a free form in the cytoplasm.

There is multiple evidence linking the inhibition of DNA topoisomerases I and II with the cytotoxic effects of antitumor drugs such as camptothecin and the DNA intercalators, 4-(9-acridinylamino)methanesulfon-m-anisidine) (mAMSA) and Adriamycin. We have assessed the effect of the DNA intercalator 3-nitrobenzothiazolo(3,2-a)quinolinium (NBQ) on the DNA topoisomerase I and II activities. NBQ has no effect on the activity of purified topoisomerase I, whereas it induced purified topoisomerase II binding to DNA without inducing DNA scission. Above 30 microM, NBQ stimulated formation of double- and single-strand breaks mediated by topoisomerase II in plasmid DNA. Using the alkaline elution technique we determined that NBQ induced single-strand and DNA-protein-associated breaks in the human promyelocytic leukemia cell line HL-60. At sublethal concentrations (less than or equal to 1 microM), NBQ induce HL-60 cells to differentiate. Topoisomerase II-mediated DNA cleavage induced by mAMSA was substantially reduced in NBQ-differentiated cells. Our data suggest that topoisomerase II could play a major role in the biological activity of NBQ in vivo.

In an attempt to get an insight into the activity of mAMSA (a DNA topoisomerase II-mediated drug) on the human proto-oncogene c-myc, an in vitro system consisting of purified calf thymus DNA topoisomerase II and a c-myc DNA inserted in lambda phage was utilized. The occurrence of discrete bands, detected by hybridization of Southern blots with appropriate c-myc probes, indicated the presence of cleavage sites in the sole presence of DNA topoisomerase II. The band intensity increased in the presence of mAMSA, while no significant difference occurred in the cleavage pattern. The location of the cleavage sites along the c-myc locus revealed a striking correspondence with that of some DNase hypersensitive sites. These results indicate that DNA topoisomerase II is most certainly implicated in the mAMSA activity and that the drug stimulates the topoisomerase II cleaving activity at specific sites, which may be involved in the biological activity of the drug.

A novel DNA-topoisomerase from Trypanosoma cruzi was partially purified. The enzyme, without ATP addition, catalyzes decatenation of kinetoplast DNA, catenation of circular supercoiled DNAs and unknotting of P4 phage DNA. The presence of Mg++ is required as well as a suitable concentration of KCl. In stoichiometric conditions the trypanosome enzyme induces double-strand DNA cleavage. The reaction is highly stimulated by some chemicals. Such characteristics allow to include this enzyme into the type II class of DNA-topoisomerases.

The antitumor drugs mAMSA and VM26 were shown to stimulate the topoisomerase II (Topo II) cleavage activity on the c-myc protooncogene in several human tumor cell lines (N417, HL60, EJ, H146, CaSki, A431, IGROV1, and CAL18A) and human peripheral lymphocytes. The mAMSA-induced gene cleavage was found to increase with the steady-state levels of c-myc transcripts in cell lines while no cleavage could be evidenced in the other genes so far tested. In mAMSA-treated N417 cells, the overall genomic DNA cleavage detected by alkaline elution was found to be about 20 times lower than the c-myc gene cleavage. Topo II mRNA levels were associated with the nuclear Topo II decatenating activity in cell lines and increased with c-myc cleavage. Topo II decatenating activity was found to be 3 times lower in quiescent than in exponentially growing N417 cells, but the c-myc cleavage induced by mAMSA was found as intense in quiescent as in growing cells. Thus, our data seem to indicate that c-myc gene cleavage is not related to cellular Topo II content but rather to c-myc gene transcription. Therefore, we suggest that only a small fraction of the Topo II is able to react with drug on the c-myc gene in relation to its transcriptional accessibility. Since c-myc overexpression is frequently found to be related to human cancer progression, we suggest that this gene could be an important target for Topo II related antitumor drugs.