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PubMedDate not supplied

Interaction of intercalating and non-intercalating agents with DNA: use of hydroxyapatite chromatography and S1 nuclease.

We have used hydroxyapatite (HA) chromatography and S1 nuclease hydrolysis to study the modification in the secondary structure of DNA caused by certain intercalating and non-intercalating ligands. The principal conclusions of HA experiments were as follows: (1) when native DNA, complexed with drugs believed to bind to DNA by intercalation (ethidium bromide, acridine orange, actinomycin D and acriflavin), is chromatographed on HA a lower affinity of DNA for HA is observed; also, the DNA elutes from HA columns as a drug-DNA complex; (ii) ligands that are known to interact with DNA by surface interactions do not show these effects; (iii) it may be possible to quantitate the binding of the intercalating drug to DNA and to determine its degree of binding by HA chromatography. Possibly, intercalation causes a change in the configuration of the sugarphosphate backbone of DNA, resulting in an altered steric orientation or 'burial' of phosphate groups with reduced availability for surface interactions with HA. S1 nuclease was used to determine the thermal melting profiles of DNA complexed with ethidium bromide and acridine orange. The melting profile in both cases was found to be biphasic with considerably reduced denaturation even at 95 degrees C. This is accounted for by the property of intercalating agents of stabilizing the secondary structure of DNA and the reported preference in binding to G-C base pairs.

Animals↗
PubMed1992

Molecular analysis of mutations induced by the intercalating agent ellipticine at the hisD3052 allele of Salmonella typhimurium TA98.

We have used DNA colony hybridization, the polymerase chain reaction (PCR), and direct DNA sequencing to determine the mutations induced by the intercalating agent ellipticine in Salmonella typhimurium TA98 in the presence of S9. Of 400 ellipticine-induced revertants that were selected at a mutant yield that was ninefold over the background, 85.5% contained a GC or CG deletion within a common CGCGCGCG hotspot; this deletion occurred among 47% of the spontaneous revertants. In addition to this hotspot, the ellipticine spectrum contained two deletion warmspots that reside opposite each other in looped-out regions of a possible DNA secondary structure. Ellipticine and its metabolites likely revert Salmonella strain TA98 by forming DNA adducts that promote slippage-mismatches and by stabilizing these slipped mismatched sequences via intercalation. The involvement of these mechanisms, along with a likely role for DNA secondary structures and a possible role for DNA gyrase, may account for the site specificity exhibited by ellipticine in strain TA98.

Base Sequence↗
PubMed1994

The action of the DNA intercalating agents 4'-(9-acridinylamino) methanesulphon-m-anisidide and 1,4-bis(butylamino) benzo[g]phthalazine in U-937 human promonocytic cells: relationship between cell cycle and differentiation.

The action of two structurally related DNA intercalating agents has been studied and compared, namely 4'-(9-acridinylamino) methanesulphon-m-anisidide (amsacrine, mAMSA) and 1,4-bis(butylamino)benzo[g]phthalazine (ABP) on the cell cycle and differentiation of U-937 human promonocytic leukemia cells. mAMSA (0.1 microM) and ABP (4 microM) reduced the proliferation activity to a similar extent and caused little cell mortality. At these subcytotoxic concentrations mAMSA induced the cells to accumulate at the G2 phase of the cycle, while cycle inhibition provoked by ABP was not phase specific. In addition, mAMSA caused an increase in the cell mass while ABP provoked cell shrinkage. This was consistent with the fact that ABP considerably inhibited protein synthesis, while mAMSA did not significantly affect this activity. SDS/K+DNA precipitation assays indicated that mAMSA, but not ABP, stimulated protein-DNA covalent complex formation. Finally, it was found that mAMSA, but not ABP, elicited the expression of differentiation markers, namely nitroblue tetrazolium reduction, activation of vimentin and leukocyte integrin (CD11b/CD18 and CD11c/CD18) expression, and downregulation of c-myc expression. The DNA intercalators doxorubicin and mitoxantrone, which like mAMSA induced the cells to accumulate at the G2 phase and increased the cell mass, induced the expression of differentiation markers. In contrast, the intercalators aclarubicin and caffeine and the non-intercalator novobiocin, which produced minor alterations on cell-cycle distribution and caused cell shrinkage, did not significantly elicit differentiation. These results support the conclusion that differentiation of myeloid leukemia cells by cytostatic drugs depends on the perturbations of the cell cycle, leading to disproportionate increases in cell mass.

Amsacrine↗
PubMed1991

Induction of DNA bending by bifunctional intercalating agents of the 7H-pyridocarbazole family.

The structures and binding energetics of selected complexes formed between the deoxynucleotides d(CpGpGpCpG).d(CpGpCpCpG), d(CpGpApTpCpG)2, d(GpCpGpCpCpG).d(CpGpGpCpGpC), and d(CpGpCpCpCpG)2 with the DNA bifunctional intercalating agent ditercalinium and three of its rigid linking chain derivatives have been investigated theoretically by means of a molecular mechanics approach that takes into account nucleic acid flexibility, ligand flexibility and solvent dielectric effects (R. Lavery, in: Unusual DNA structures, eds S. Harvey and R. Wells (Pergamon, New York, 1988) p. 189; R. Lavery, in: DNA bending and curvature, eds W.K. Olson et al. (Adenine Press, New York, 1988) p. 191). The piperidinium chains of the bis-intercalating ligands are always located in the major groove of DNA. For the energy-minimized complexes the ligand proceeds to bind following preferentially the 5'-pyrimidine-purine-3' alternating sequence, thus dictating the number of internal exclusion sites. The complexes with three exclusion sites will present (i) a bending of the structure towards the major groove, and (ii) a non-ideal distribution of unwinding angles; complexes with less than three exclusion sites will remain essentially linear. The absence of a bend does not preclude other types of local deformations of the base-pairs such as inclination, buckle and tip. The proposed structures of the d(CpGpApTpCpG)2 complexes are in agreement with NMR structural results. The possible relevance of these findings to a previously proposed mode of interaction for ditercalinium-like DNA ligands is discussed.

DNA↗
PubMed1983

Failure of the intercalating agent 4'-(9-acridylamino)-methanesulphon-m -anisidide to induce DNA-repair replication in cultured human cells.

A search has been made for DNA-repair replication in cultured human cells treated with the DNA-intercalating agent 4'-(9-acridinylamino) methanesulphon--m-anisidide (m-AMSA). Previous reports had suggested that the transient appearance of protein-associated DNA-strand breaks in mammalian cells treated with m-AMSA might be indicative of a rapid DNA-repair process. The present experiments suggest that such a repair process is unlikely to occur as, even in cells treated with high concentrations of m-AMSA (50 microM) for 20 h, DNA repair replication could not be detected down to the limits of sensitivity (1 million nucleotides replaced per cell).

Aminoacridines↗
PubMedDate not supplied

Mutagenicity of some intercalating agents in Chinese hamster V79 cells.

ICR 170 and ICR 191, but not 9-aminoacridine or chloroquine, induced both 6-thioguanine- and, to a smaller extent, ouabain-resistance in Chinese hamster V79 cells. These results indicate that covalent binding to DNA is necessary for intercalating agents to induce mutation in this cell line, and that this assay can distinguish potential carcinogens from non-carcinogenic analogues of this chemical type. The induction of ouabain-resistance by both ICR 170 and ICR 191 indicates that these frameshift mutagens induce base-pair substitution to some extent in V79 cells.

Animals↗
PubMed1985

Anthrapyrazoles, a new class of intercalating agents with high-level, broad spectrum activity against murine tumors.

A series of 5-[(aminoalkyl)amino]-substituted anthra[1,9-cd] pyrazol-6(2H)ones (anthrapyrazoles) were synthesized. These compounds, which differ from the anthracenediones in that an additional pyrazole ring has been fused to the anthracene system in place of one carbonyl group, were evaluated in vivo for their anticancer activity in eight different mouse tumor systems. Compounds were selected for testing primarily on the basis of their high levels of activity P388 leukemia and occasionally for structural considerations. Sixty-seven % of the 21 analogues studied were curative in the National Cancer Institute P388 screen. Many of the compounds tested were highly active against each of the tumors of the National Cancer Institute panel. Thus 82, 73, 45, and 80% of the compounds tested were curative for L1210 leukemia, B16 melanoma, M5076 sarcoma, and the MX-1 mammary xenograft, respectively. Several of the compounds studied were curative against every tumor of the above panel. Because of the high activity of the anthrapyrazole series as a class in the National Cancer Institute tumor panel, additional testing was necessary to allow selection of clinical candidates. Twenty-one anthrapyrazoles were tested against mammary adenocarcinoma 16C, colon adenocarcinoma 11a, and the Ridgway osteogenic sarcoma. Four compounds, PD 113,309 (Cl-937), PD 113,785 (Cl-941), PD 111,815 (Cl-942), and PD 115,593, were judged superior to the rest on the basis of the expanded panel testing. The preclinical data to date suggest that these anthrapyrazoles are similar to doxorubicin in both degree and spectrum of activity. Each of these anthrapyrazoles were significantly more active than were the other synthetic intercalating agents, the anthracenediones mitoxantrone and ametantrone, against the tumors of the expanded panel. On the basis of their high level of broad spectrum activity in preclinical systems, ease of formulation, possible lack of cross-resistance with doxorubicin, and potential lack of cardiotoxicity, Cl-937, Cl-941, and Cl-942 have been selected for further preclinical evaluation and possible clinical development.

Animals↗
PubMed1980

Simultaneous detection of chromosomal aberrations and sister-chromatid exchanges: experience with DNA intercalating agents.

Micronuclei that arise from chromosomal fragments can be used as an index of cytogenetic damage in a number of mammalian cell-culture systems. Since sister-chromatid exchanges (SCE) can be scored at the second metaphase after treatment, micronuclei that have arisen from acentric fragments at the first mitosis should be present in the same preparations. When a series of 8 mutagens, most of them intercalating agents, was studied in Chinese hamster ovary (CHO) cells, increased frequencies of micronuclei were detected at all doses which induced increased frequencies of SCE. The time required to score the slides for micronuclei was about one tenth that required for SCE. Since it is clear that aberrations and SCE have different genetic consequences and that they arise by mechanisms that differ at least in part, we find it useful to be able to measure both on the same slides. In this way mutagenic agents such as X-rays and bleomycin that produce few, if any, SCE but much chromosomal breakage would not be missed, nor would any agent that produced SCE without aberrations.

Animals↗
PubMed1984

Denaturation of nucleic acids induced by intercalating agents. Biochemical and biophysical properties of acridine orange-DNA complexes.

At high binding densities acridine orange (AO) forms complexes with ds DNA which are insoluble in aqueous media. These complexes are characterized by high red- and minimal green-luminescence, 1:1 (dye/P) stoichiometry and resemble complexes of AO with ss nucleic acids. Formation of these complexes can be conveniently monitored by light scatter measurements. Light scattering properties of these complexes are believed to result from the condensation of nucleic acids induced by the cationic, intercalating ligands. The spectral and thermodynamic data provide evidence that AO (and other intercalating agents) induces denaturation of ds nucleic acids; the driving force of the denaturation is high affinity and cooperativity of binding of these ligands to ss nucleic acids. The denaturing effects of AO, adriamycin and ellipticine were confirmed by biochemical studies on accessibility of DNA bases (in complexes with these ligands) to the external probes. The denaturing properties of AO vary depending on the primary structure (sugar- and base-composition) of nucleic acids.

Acridine Orange↗
PubMed1987

Oligo(alpha-deoxynucleotide)s covalently linked to intercalating agents: differential binding to ribo- and deoxyribopolynucleotides and stability towards nuclease digestion.

An octathymidylate was synthesized with the alpha anomer of thymidine instead of the naturally occurring beta anomer. This oligonucleotide binds to complementary sequences containing beta-nucleosides. Binding to ribose-containing oligomers and polymers is much stronger than binding to deoxyribose-containing analogs. A derivative of acridine (9-amino-6-chloro-2-methoxyacridine) was covalently attached either to the 5' phosphate or to the 3' phosphate of the alpha-octathymidylate. A pentamethylene linker was used to bridge the phosphate group and the 9-amino group of the acridine derivative. In both cases the complexes with the complementary sequences were strongly stabilized due to the additional binding energy provided by intercalation of the acridine ring within the miniduplex structure formed by the oligonucleotide with its target sequence. The acridine-substituted alpha-oligothymidylates did not lose their discrimination between ribose and deoxyribose-containing complementary sequences. The alpha-oligothymidylates were much more resistant towards endonucleases than their beta analogs, independently of whether they were linked to the acridine derivative. Acridine substitution provided additional protection against the corresponding exonucleases. alpha-Oligodeoxynucleotides covalently linked or not to intercalating agents represent families of molecules that open possibilities to block mRNA translation or viral RNA expression in vitro and in vivo.

Aminoacridines↗
PubMed1987

Oligo-[alpha]-deoxynucleotides covalently linked to an intercalating agent. Double helices with parallel strands are formed with complementary oligo-[beta]-deoxynucleotides.

An oligo-[alpha]-deoxynucleotide of sequence (5')d(TCTAAACTC) (3') was synthesized using the alpha-anomers of deoxynucleosides and its 5'-phosphate was covalently linked to a 9-amino acridine derivative via a pentamethylene linker. Two oligo-[beta]-deoxynucleotides containing the complementary sequence in either the 5'----3' or the 3'----5' orientation were synthesized using natural [beta]-deoxynucleosides. Complex formation was investigated by absorption and fluorescence spectroscopies. No change in spectroscopic properties was detected with the anti-parallel [beta] sequence. Absorption changes were induced in the visible absorption band of the acridine derivative at 2 degrees C when the acridine-substituted oligo-[alpha]-deoxynucleotide was mixed in equimolecular amounts with the complementary [beta]-sequence in the parallel orientation. Hypochromism was observed in the UV range. The fluorescence of the acridine derivative was quenched by the guanine base present in the second position of the complementary sequence. Cooperative dissociation curves were observed and identical values of melting temperatures were obtained by absorption and fluorescence. An increase in salt concentration stabilized the complex with a delta Tm of 8 degrees C when NaCl concentration increased from 0.1 to 1 M. These results demonstrate that an oligo-[alpha]-deoxynucleotide covalently linked to an intercalating agent is able to form a double helix with an oligo-[beta]-deoxynucleotide. The two strands of this [alpha]-[beta] double helix adopt a parallel 5'----3' orientation. The acridine ring is able to intercalate between the first two base pairs on the 5'-side of the duplex structure.

Base Sequence↗
PubMed1988

Selective loss of mitochondrial DNA after treatment of cells with ditercalinium (NSC 335153), an antitumor bis-intercalating agent.

Ditercalinium (NSC 335153), a bifunctional intercalating molecule with antitumor activity, is found to express its toxicity through a mechanism of action completely different from that of other monointercalating agents. Electron microscopic observation of ditercalinium-treated cells shows a drastic alteration of mitochondrial structure. Cells deficient in mitochondrial respiration (GSK3 cells) isolated by A. Franchi et al. (Int. J. Cancer, 27: 819-827, 1981) are about 25-fold more resistant than cells deficient in glycolysis (DS7 cells) isolated by J. Pouysségur et al. (Proc. Natl. Acad. Sci. USA, 77: 2698-2701, 1980). Revertants have been isolated from GSK3 cells. In these cells, the sensitivity to ditercalinium has been recovered with mitochondrial respiration. Ditercalinium treatment of L1210 leukemic mouse cells leads to a specific elimination of mitochondrial DNA detected by DNA-DNA hybridization. No measurable alteration of nuclear DNA is observed. In contrast, the monomeric analogue of ditercalinium only alters nuclear DNA and does not change the mitochondrial DNA content. The activity of cytochrome c oxidase, an enzyme which contains a subunit coded by the mitochondrial DNA, decreases exponentially in treated cells with a half-life of 24 h, corresponding to the turnover of the enzyme. These results suggest that ditercalinium exerts a specific cytotoxic effect at the level of mitochondrial DNA. This action could account for the delayed cytotoxicity induced by this compound.

Animals↗
PubMed1992

Dissociation of cytotoxicity and DNA cleavage activity induced by topoisomerase II-reactive intercalating agents in hamster-human somatic cell hybrids.

Previous studies using the mutant Chinese hamster ovary cell line VpmR-5 indicate that its resistance to epipodophyllotoxins and intercalating agents is likely to be mediated through a qualitative change in type II topoisomerase that confers resistance to drug-stimulated DNA cleavage activity. In a further investigation of the genetic basis of drug resistance in VpmR-5 cells, we fused a hypoxanthine-guanine phosphoribosyl transferase-deficient subline of VpmR-5 (Vtgm-6) with normal human lymphocytes and analyzed the resultant hybrid lines (HL) for altered drug sensitivity. In all, 3 of 16 hybrid clones exhibited partial reconstitution of sensitivity to etoposide, mitoxantrone, doxorubicin, and 5-iminodaunorubicin while retaining complete resistance to m-AMSA. However, enhanced sensitivity to drug-induced DNA cleavage activity was observed only for etoposide. Biochemical and molecular-marker analysis of the hybrids failed to identify human chromosome 17 (the provisional location of TOP2) or any other human chromosome that is consistently and uniquely associated with drug sensitivity. We therefore sought to verify the chromosomal assignment of TOP2 by Southern blot hybridization of TOP2 cDNA on a human hybrid mapping panel and confirmed its location on chromosome 17. However, no hybridizing sequence to the TOP2 cDNA was found in any of the 16 Vtgm-6 hybrid lines. Efforts to select more directly for human chromosome 17 VpmR-5 hybrids using microcell fusion of mouse A9 cells carrying human 17 linked to pSV2neo were unsuccessful. None of the five hybrid clones thus obtained had 17q markers, including the gene for TOP2. Although the mechanism underlying partial reversion to a drug-sensitive phenotype in the original Vtgm-6 hybrid lines has yet to be defined, the data obtained in these lines indicate that anthracycline- and anthracenedione-induced cytotoxic effects can be dissociated from DNA cleavage activity. This suggests that pathways distal to cleavable-complex formation or, alternatively, independent of interactions with topoisomerase II that involve other intracellular targets are important in mediating the cytotoxicity produced by these drugs.

Animals↗
PubMed1998

High-resolution capillary electrophoretic separation of supercoiled plasmid DNAs and their conformers in dilute hydroxypropylmethyl cellulose solutions containing no intercalating agent.

The three conformers of plasmid pBR322, linear, supercoiled and nicked circular forms, were separated by capillary electrophoresis (CE) in 0.1% hydroxypropylmethyl cellulose (HPMC) solution in the absence of intercalating agents and the migration order was confirmed by co-migration of enzymatically prepared corresponding DNAs. The previously observed broad peaks of supercoiled DNAs in CE are results of unresolved peaks of topoisomers which differ only in the degrees of twisting. We have demonstrated the separation of an artificial topoisomer ladder made from pBR322 and topoisomerase I. The population of topoisomers of a supercoiled DNA is dependent on sample matrices and separation conditions.

DNA Topoisomerases, Type I↗
PubMed1976

Mechanisms and selectivity of anthracycline aminoglycosides and other intercalating agents.

Many of the antiproliferative actions of daunorubicin and adriamycin are attributable to the results of their interaction with DNA: single and double strand breaks and inhibition of repair and of nucleic acid synthesis. Like other DNA-reactive agents, high cumulative doses produce pharmacological actions and species-specific cytotoxicities in non-proliferating myocardium and nerve cells. Evidence is reviewed suggesting that many, if not all, intercalating agents are membrane-reactive. Thus it seems likely that the selective nature of the responses by various cells and the discrete expressions of cytotoxicity may be determined by binding to specific receptors in membranes of target cells in susceptible organs and species.

Adenosine Triphosphatases↗
PubMed2002

Putative identification of functional interactions between DNA intercalating agents and topoisomerase II using the V79 in vitro micronucleus assay.

Clastogenicity is frequently observed following treatment of mammalian cells with new chemical entities. This clastogenicity, unless proven otherwise, is assumed to result from the imperfect repair of DNA lesions produced from covalent chemical/DNA interaction. However, clastogenicity can also arise via other mechanisms such as non-covalent chemical intercalation into DNA resulting in poisoning of cellular DNA topoisomerase II (topo II) and stabilization of DNA double strand breaks. We have recently reported modifications to the V79 in vitro micronucleus assay which allow an indirect evaluation of both the intercalative and topoisomerase-interactive activities of chemical agents. In the present studies we have used these modified assays to further assess the validity of this approach in an evaluation of a number of intercalating and non-intercalating polycyclic compounds. It is shown that intercalating agents may be catalytic topo II inhibitors (e.g. chloroquine (CHL), tacrine (TAC), 9-aminoacridine (9AA), ethidium bromide (EB)) or topo II poisons (e.g. proflavine (PROF), auramine O (AUR) and curcumin (CURC)). Still other intercalators are shown to lack detectable topo II-interactions, (e.g. imipramine (IMP), quinacrine (QUIN), 2-aminoanthracene (AA), iminostilbene (IMN) and promethazine (PHE)). It is concluded that (1) the clastogenicity of three agents, PROF (a typical DNA intercalating agent), and AUR and CURC (both structurally atypical intercalating agents, with unknown clastogenic mechanisms), may be due to topo II poisoning; (2) other intercalating agents may either act as catalytic topo II inhibitors or exhibit no functional topo II interaction; (3) The use of these cell-based approaches may provide a logical first step in determining if unexpected clastogenicity associated with test article exposure is due to a topo II interaction.

Animals↗