ABSTRACT
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.
Subject(s)
Anthraquinones/pharmacology , DNA, Superhelical/chemistry , DNA, Superhelical/drug effects , DNA/chemistry , DNA/drug effects , DNA, B-Form/chemistry , DNA, B-Form/drug effects , Denaturing Gradient Gel Electrophoresis , Intercalating Agents/pharmacology , Microscopy, Atomic Force , Nucleic Acid Conformation/drug effects , Plasmids/chemistry , Plasmids/drug effects , Spectrometry, FluorescenceABSTRACT
Five novel proflavine-dithiazolidinone derivatives 4a-4e have been designed and synthesized by the reaction of dialkyl acridin-3,6-diyl dithioureas 3a-3e with methyl bromoacetate. The binding affinity of dithiazolidinone hydrochlorides 5a-5e with calf thymus DNA and plasmid (pUC19) DNA was investigated by a variety of spectroscopic techniques including UV-vis, fluorescence, and CD spectroscopy. The effects of 5a-5e on the thermal denaturation profiles of calf thymus DNA were also studied. From spectrophotometric and spectrofluorimetric titrations, the binding constants for the pUC19 DNA-drug complexes were determined (K = 6.2-2.2 x 104 M-1). In vitro cytotoxic activities of compounds 5a-5e toward murine leukemia cell line L1210 and human uterus carcinoma HeLa cells were also examined. 2',2' '-[(Acridin-3,6-diyl)diimino]-3',3' '-dipropyl-1,3-dithiazolidin-4-one hydrochloride (5b) showed the highest activity against these cells with IC50 values of 6.3 microM and 12.9 microM over the course of 72 h.
Subject(s)
Acridines/chemistry , DNA/chemistry , Proflavine/chemical synthesis , Proflavine/toxicity , Thiazoles/chemistry , Animals , Cattle , Cell Line, Tumor , Cell Shape , Cell Survival/drug effects , Electrons , Humans , Mice , Molecular Structure , Nucleic Acid Denaturation , Photochemistry , Proflavine/chemistry , Spectrum Analysis , Titrimetry , Transition TemperatureABSTRACT
Temperature-gradient gel electrophoresis (TGGE) was used to study DNA-drug interactions. The results indicate that at least two classes of DNA intercalating drugs are distinguishable with respect to temperature increase: reversible and irreversible. The method offers an excellent means of visualizing the melting profile of an individual DNA topoisomer in the presence of DNA binding drugs. Our findings coincide with UV/VIS absorption spectroscopy data.
Subject(s)
DNA/analysis , DNA/drug effects , Electrophoresis, Agar Gel/methods , Intercalating Agents/pharmacology , Biophysical Phenomena , Biophysics , DNA/chemistry , Nucleic Acid Conformation , Plasmids/analysis , Plasmids/chemistry , Plasmids/drug effects , Spectrophotometry , TemperatureABSTRACT
The conformational stability of individual DNA topoisomers depends on the concentration of DNA intercalating drugs. To study the DNA-drug interaction, we used ethidium bromide (EtBr) and negative supercoiled pUC19 as a model system. The effects of two anthracyclines widely used in cancer therapy, daunorubicin (Dau) and doxorubicin (Doxo), and EtBr were compared. In spite of their different chemical structures and intercalation mode, all intercalating agents show similar effects on the conformational stability of supercoiled DNA. Our observations show that the studied intercalators have at least two main effects on the supercoiled DNA: (i) they decrease the level of negative supercoiling and, at certain concentrations, they may induce positive supercoiling in DNA; (ii) a temperature increase can cause a recovery of negative supercoiling in DNA. The conformational stability of plasmid DNA-drug complexes has been investigated by temperature gradient gel electrophoresis (TGGE). We demonstrate the suitability of TGGE for this purpose, because it offers a global view on DNA-drug complexes over a continuous range of temperature. Images of DNA plasmids adsorbed onto a substrate at different temperatures and drug concentrations were acquired by atomic force microscopy (AFM), allowing us to distinguish directly the conformation chirality assumed by the plasmid under different conditions confirming TGGE results. Our detection system allows to characterize unknown drugs and to determine their intercalating properties.
Subject(s)
Anthracyclines/pharmacology , DNA, Superhelical/chemistry , Hot Temperature , Bacteriophages/genetics , DNA, Superhelical/drug effects , DNA, Superhelical/radiation effects , Daunorubicin/pharmacology , Doxorubicin/pharmacology , Electrophoresis, Agar Gel/methods , Ethidium/pharmacology , Intercalating Agents/pharmacology , Microscopy, Atomic Force , Nucleic Acid Conformation/drug effects , Nucleic Acid Conformation/radiation effects , Phase Transition/drug effects , Phase Transition/radiation effectsABSTRACT
Changes in DNA supercoiling might be essential to generate the response of cellular machinery to temperature stress. The heat-induced structural transition for a topoisomer depends on the value of its specific linking difference. We detect only less negatively supercoiled DNA and an abundance of alternative irregular DNA forms at culture temperatures close to the growth limit of Escherichia coli. We show that the irregular forms are derived from regular plasmid DNAs and their population in the cells is temperature-dependent. Here, we show that it is possible to isolate and characterize individual DNA topoisomers directly from cells without a topoisomerase treatment. Temperature gradient gel electrophoresis (TGGE) and atomic force microscopy (AFM) were used to study the effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability.
