抗癌药ADM与DNA嵌插复合物的模型化研究

通过对高效抗癌药阿霉素(Adriamycin)与三碱基片段理论DNA受体的嵌插相互作用的快速能量最小化计算,其中包括阿霉素以三种嵌插方式,与两种核酸受体(d(C—GC)和d(T—AT)的相互作用,以理论上论证了大沟平行嵌入方式,即Pigram-Fuller-Hamilton模型为能量最优模型,讨论了非嵌插相互作用在ADM—DNA(3bp)体系最优构象选择上的主导作用,同时,说明了阿霉素的嵌插选择性依赖于嵌插方式.     

Topography of nucleic acid helices in solutions. XXIV. Intercalation specificities of DNA and their possible role in the recognition process

Through the use of reporter molecules, I, it is shown that more than one type of intercalating site must exist in DNA. Although this finding is completely consistent with the DNA structure it has not yet been demonstrated. The significance of the presence of different intercalating sites in DNA may be of extreme importance in the recognition of nucleic acid-protein systems.     

Frameshift mutagenesis in Salmonella typhimurium by reversible DNA intercalators: effect of a UVR B mutation

The simple reversible intercalating agents isopropyl-oxazolopyridocarbazole and 9-aminoacridine have been found to induce frameshift -1 mutations at a much lower level in Salmonella typhimurium delta uvrB TA 1537 than in the uvr+ wild type TA 1977 strain. This phenomenon can neither be explained by differential cytotoxicity of the drug nor by selective permeation and accessibility to intercalating sites to bacterial DNA. These finding indicate that the lower mutagenicity of intercalating agents in the delta uvrB strains does not result from nonspecific phenotypic modifications of parameters which control the mutagenesis. That leads to the hypothesis that in agreement with the Streisinger's model, the excision repair system could be directly involved in the appearance of frameshift mutations.     

Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.     

Synthesis and hybridization properties of oligonucleotides containing 2'-O-modified ribonucleotides.

A versatile, general way is described for the introduction of different functional groups into oligonucleotides by means of a simple linker at the 2'-position of the sugar. Nucleotide building blocks carrying lipophilic, intercalating or tertiary amino groups can be placed deliberately at any desired position of oligonucleotides by standard automated oligonucleotide synthesis. Thermal denaturation studies with these oligonucleotides reveal the following general trends: i) Modification with lipophilic n-octyl groups has little if any effect on duplex stability; a destabilizing (lipophilic) substituent is better tolerated at or near the ends than in the middle of the oligo. ii) An intercalating substituent (2-aminoanthraquinone) substantially increases duplex stability. iii) N,N-Dimethyl amino residues also increase duplex stability though to a smaller extent than intercalating residues. iv) Modifications at the 5'-end have a more pronounced influence on the TM than the corresponding 3'-modifications. v) Oligonucleotides modified in such a way show little or no loss in sequence specificity.     

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.     

Condensation of DNA in situ in metaphase chromosomes induced by intercalating ligands and its relationship to chromosome banding

Interactions of certain intercalating cationic ligands with nucleic acids result in the formation of products that undergo condensation and agglomeration; this transition in solution can be monitored by light-scatter measurements. In the present study, using such intercalators as the antitumor drug mitoxantrone or fluorochromes acridine orange and quinacrine, we induced condensation of DNA in situ in Chinese hamster chromosomes. The in situ products scattered light and could be detected by darkfield- or phase-contrast microscopy. In the darkfield the complexes had a characteristic granular appearance and often generated a banding pattern on the chromosomes. In contrast, condensation of DNA in situ by the nonintercalating polyvalent cations (Co3+, spermine4+), while enhancing the chromosome's image contrast, did not produce the granular products or the banding. The condensation of free DNA, single or double stranded, natural or synthetic, the latter of various base composition and configuration, was also measured in solution. The condensation in solution and in situ was observed at similar concentrations of the respective ligands. The intercalating dye ethidium bromide, which did not condense DNA in solutions of moderate and high ionic strength, also did not generate the granular products or banding on chromosomes. The data also show that both base composition and configuration are important factors in determining the sensitivity of DNA to condensation by particular intercalating ligands. The studies suggest that the phenomenon of DNA condensation by intercalating dyes, which shows a high degree of specificity with respect to primary and secondary structures of DNA, may be associated with mechanisms of chromosome banding induced by the intercalating thiazine dyes in Giemsa staining or by quinacrine. Observation of chromosome banding based on light-scatter detection in darkfield microscopy allows the study of interactions between DNA and the ligands that neither fluoresce nor generate colored products. This principle of chromosome "counter-staining" can be explored by flow cytometry.     

Relationship between cytostatic activity of oxazolopyridocarbazoles and accessibility of DNA intercalation sites in living bacteria

The ability of oxazolopyridocarbazole (OPC) derivatives to interact with DNA in living bacteria through reversible intercalation has been determined by using as probes their selective mutagenic effect on Salmonella typhimurium TA 1977 and TA 1537 as detected by frame-shift-1 reversion, the absence of intervention of the error-prone repair system on the mutagenic efficiency, the absence of induction of the SOS functions, and the absence of effect of recA and uvrB mutations on their bacteriostatic properties. Involvement of simple reversible intercalation as the event responsible for the bacteriostatic effect of the drugs has been further investigated by the establishment of a significant correlation between the maximum number of accessible intercalating sites in living bacteria and the bacteriostatic effect expressed in terms of the ED50. This correlation has been established by using bacteria spontaneously exhibiting different sensitivities toward the drugs as well as a resistant strain obtained by adaptation in the presence of increasing amounts of isopropyl-OPC. The number of intercalating sites in living bacteria was determined by using the change in the fluorescence properties of the drugs upon binding to intercalating sites. The results obtained clearly demonstrate that the number of intercalating sites is the parameter that controls the bacteriostatic effect of the drugs, indicating that DNA is the target for these drugs and that reversible intercalation is responsible for the cytostatic effect.     

A Mechanism for the Entrapment of DNA at an Air-Water Interface

Addition of the intercalating dye quinacrine to a low ionic strength solution of DNA in quantities sufficient to saturate the high affinity sites in the DNA will result in the accumulation of the DNA at the solution interface. This entrapment of DNA at the air-water interface has been assayed by the adsorption of DNA to untreated carbon-coated electron microscope grids touched to the solution surface. Other intercalating dyes can also bring about this entrapment, if they possess a side arm large enough to occupy one of the DNA grooves when the dye is intercalated into the DNA. The extension and unwinding of the DNA helix brought about by the intercalating chromophore of the dye molecules are not requirements for the entrapment process. Spermidine, a simple polyamine that will bind to the DNA minor groove but that has no intercalating chromophore, was found to bring about this entrapment. Even simple mono- and divalent cations in the absence of the above ligands were found to promote a low level of surface entrapment. A model for the entrapment of DNA at the air-water interface is proposed in which one (or both) of the hydrophobic grooves of the DNA becomes a surface-active agent as a consequence of the association of various ligands and charge neutralization.     

Low concentrations of acridine dimers inhibit micrococcus AP endonuclease through interaction with apurinic sites in DNA.

The effect of dimeric DNA intercalating compounds was assayed on a purified AP endonuclease from Microccoccus luteus using apurinic supercoiled PM2 DNA as a substrate. Binding on apurinic sites was estimated through the competition with the intercalating compound, 9-NH2-ellipticine, which displays great specificity for apurinic sites. An acridine dimer with a spermine linker is at 0.1 microM the best inhibitor of cleavage at the apurinic site induced either by the AP endonuclease or by 9-NH2-ellipticine. Bisintercalating agents are more effective inhibitors of AP endonuclease than monointercalating ones. Most effective inhibitors among dimers have acridine residues.     

Effect of alkaloids from celandine on calcium accumulation and oxidative phosphorylation in mitochondria depending on their DNA intercalating properties

The effect of principal alkaloids (sanguinarine, chelerythrine, coptisine, chelidonine) of greater celandine Chelidonium majus L., as well as the alkaloids from Colchicum autumnale L. (colchicine and colchamine) on calcium accumulation and oxidative phosphorylation in rat liver mitochondria has been studied. The obtained data were compared with DNA intercalating properties of alkaloids detected by the method of thermodenaturation (DNA melting curve plots). It was found that chelerythrine and sanguinarine blocked absorption and accumulation of calcium cations and inhibited oxidative phosphorylation, while the coptisine significantly diminished those indices. Chelidonine, colchicines and colchamine had no influence on the studied characteristics. The effect of alkaloids upon mitochondria functional state correlated tightly with their DNA intercalating properties: chelerythrine and sanguinarine were strong intercalators, while coptisine was a weak one, and chelidonine, colchicine and colchamine did not interact with DNA and caused no changes in its melting point. Correlation coefficient between the intercalating properties of alkaloids and their inhibition of calcium accumulation was 0.89, and with their oxidative phosphorylation inhibition - 0.93. It is suggested that the effect of studied alkaloids upon functional properties of mitochondria can be mediated by mtDNA.     

Testing of chloroquine and quinacrine for mutagenicity in Drosophila melanogaster

To extend the data on the mutagenic effects of intercalating agents in Drosophila melanogaster, chloroquine and quinacrine were tested for the induction of genetic damage in D. melanogaster males. Sex-linked recessive lethals and sex-chromosome loss induction were studied following treatment of adult males using a feeding technique. Our results show that both intercalating compounds increase significantly the frequency of sex-linked recessive lethals, but are unable to induce sex-chromosome loss in male germ cells under the conditions of testing.     

Nuclear topoisomerase II levels correlate with the sensitivity of mammalian cells to intercalating agents and epipodophyllotoxins

We have investigated the biochemical basis for the hypersensitivity to intercalating agents and epipodophyllotoxins of a Chinese hamster cell mutant, ADR-1. More topoisomerase II-induced DNA strand breaks are accumulated by ADR-1 than by parental CHO-K1 cells following exposure to the intercalating agent amsacrine. Levels of induced DNA strand breaks correlate with cell killing. Topoisomerase II activity is elevated in ADR-1 cells as a consequence of an increased cellular level of topoisomerase II protein. We have studied the phenotype of cell hybrids generated by fusing parental and mutant cells. The hybrid ADR-1/CHO-K1 exhibits normal levels of resistance to amsacrine and expresses the lower, parental level of topoisomerase II. These results provide additional evidence that topoisomerase II mediates the cytotoxic action of intercalating agents and epipodophyllotoxins and suggest that the intracellular level of topoisomerase II is an important determinant of cellular sensitivity to these drugs. This has implications for antitumor therapy. ADR-1 cells provide a model system for studying the effects of topoisomerase II overproduction on cell proliferation and chromosome organization.     

Ellipticine-induced protein-associated DNA breaks in isolated L1210 nuclei

DNA intercalating agents have been found to produce protein-associated DNA strand breaks in mammalian cells. As a first step towards a subcellular system for the study of this reaction, we demonstrate that the reaction can take place in isolated cell nuclei. Ellipticine induces in these nuclei DNA strand breaks and stable DNA-protein complexes. Complexes and breaks are present in equivalent amounts. DNA breaks are revealed only if protein-mediated DNA adsorption to filters is abolished. These findings make it unlikely that similar effects observed in cells in culture after treatment with intercalating agents are caused by metabolically activated drugs.     

Supercoils in human DNA.

The three-dimensional structure of a double-stranded DNA molecule may be described by distinguishing the helical turns of the DNA duplex from any superhelical turns that might be superimposed upon the duplex turns. There are characteristic changes in the hydrodynamic properties of superhelical DNA molecules when they interact with intercalating agents. The hydrodynamic properties of nuclear structures released by gently lysing human cells are changed by intercalating agents in this characteristic manner. The characteristic changes are abolished by irradiating the cells with gamma-rays but may be restored by incubating the cells at 37 degrees C after irradiation. These results are interpreted as showing that human DNA is supercoiled. A model for the structure of the chromosome is suggested.     

HMG proteins released from the chromatin following incubation of mammalian nuclei with ethidium bromide

The low-molecular-mass high-mobility group (HMG) chromosomal proteins, namely HMG-14, HMG-17, and HMG-I, which have been found in several proliferating tissues, are released following incubation of nuclei isolated from young rat thymus and from human placenta in a low ionic strength medium containing the intercalating agent ethidium bromide. The amount of HMG proteins released is drug concentration-dependent, but at very high concentrations (20-40 mM) other low- and high-molecular-mass proteins, and even histones, are released. These results suggest a very weak interaction of the HMG proteins with DNA, so that they can be easily detached from the chromatin as a consequence of the interaction of DNA with the intercalating agent.     

Novel N-(3-carboxyl-9-benzyl-carboline-1-yl)ethylamino acids: synthesis, anti-proliferation activity and two-step-course of intercalation with calf thymus DNA

To explore the intercalating mechanism of -carbolines, four novel N-(3-carboxyl-9-benzyl-carboline-1-yl)ethylamino acids [-phenylalanine (6a), -alanine (6b), -isoleucine (6c) and -glycine (6d)] were prepared here. Their in vitro anticancer activities were examined by their anti-proliferation for 5 human carcinoma cell lines. The average IC50s against 5 human carcinoma cell lines are 53.54 microM, 118.77 microM, 147.34 microM and greater than 291.63 microM for 6a, 6b, 6c and 6d, respectively. The DNA intercalating mechanism of 6a-d was approved by the comparison of the parameters and signals of UV, CD and fluorescence spectra of calf thymus DNA (CT DNA) alone and the CT DNA/6a-d system. Using fluorescence titration based kinetic analysis a two-step-course consisting of stacking and intercalating was described and the stacking was considered as the key step to the CT DNA intercalating mechanism of 6a-d. Using fluorescence titration based thermomechanical analysis, the stacking complexes of 6a-d with CT DNA were described to be formed spontaneously and to be stabilized predominantly by their hydrophobic interactions. The intercalation itself goes very fast and only has limited contribution to their anticancer activities.     

Inhibition of estrogen-receptor-DNA interaction by intercalating drugs.

Ethidium bromide, an intercalating drug, was shown to inhibit the in vitro DNA binding of the uterine estradiol-receptor complex. The inhibition was reversible, dose dependent, complete for total saturation of DNA intercalating sites by the dye, and proportional to the extent of intercalated drug. The binding of the receptor to phosphocellulose and poly(adenylic acid)-cellulose was not decreased by this drug. Similar inhibition was also obtained with 9-hydroxyellipticine. Denatured DNA was more efficient at binding the estrogen receptor than phosphocellulose or poly(adenylic acid)-cellulose but less efficient than native DNA. We conclude that the DNA binding of the estrogen receptor cannot be simply interpreted in terms of electrostatic interactions but requires a particular double-helical structure of DNA.