Drug-Related Carcinogenesis: Risk Factors and Approaches for Its Prevention.

The review summarizes the data on the role of metabolic and repair systems in the mechanisms of therapy-related carcinogenesis and the effect of their polymorphism on the cancer development risk. The carcinogenic activity of different types of drugs, from the anticancer agents to analgesics, antipyretics, immunomodulators, hormones, natural remedies, and non-cancer drugs, is described. Possible approaches for the prevention of drug-related cancer induction at the initiation and promotion stages are discussed.

Re-Examination of the Esophageal Squamous Cell Carcinoma Model in Rats Induced by N-Nitrososarcosine Ethyl Ester Precursors.

Studies of the molecular mechanisms of esophageal cancer development have to be carried out on sufficient amount of tumor material, obtained under conditions of controlled exposure to carcinogenic factors. Esophageal cancer models on laboratory animals serve an indispensable source of this material. One of these models is esophageal cancer induction in rats by N-nitroso compound precursors. Despite adequate reproduction of human esophageal cancer, this model in fact has not been used since the 1990ies. Re-examination of esophageal cancer model, induced by N-nitrososarcosine ethyl ester precursors, is carried out and its efficiency in induction of squamous cell carcinoma is confirmed.

Alterations in WNT Signaling in Leukemias.

The WNT/ß-catenin signaling pathway plays an important role in the differentiation and proliferation of hematopoietic cells. In recent years, special attention has been paid to the role of impairments in the WNT signaling pathway in pathogenesis of malignant neoplasms of the hematopoietic system. Disorders in the WNT/ß-catenin signaling in leukemias identified to date include hypersensitivity to the WNT ligands, epigenetic repression of WNT antagonists, overexpression of WNT ligands, impaired ß-catenin degradation in the cytoplasm, and changes in the activity of the TCF/Lef transcription factors. At the molecular level, these impairments involve overexpression of the FZD protein, hypermethylation of the SFRP, DKK, WiF, Sox, and CXXC gene promoters, overexpression of Lef1 and plakoglobin, mutations in GSK3ß, and ß-catenin phosphorylation by the BCR-ABL kinase. This review is devoted to the systematization of these data.

Structure, Properties, and Biological Relevance of the DNA and RNA G-Quadruplexes: Overview 50 Years after Their Discovery.

G-quadruplexes (G4s), which are known to have important roles in regulation of key biological processes in both normal and pathological cells, are the most actively studied non-canonical structures of nucleic acids. In this review, we summarize the results of studies published in recent years that change significantly scientific views on various aspects of our understanding of quadruplexes. Modern notions on the polymorphism of DNA quadruplexes, on factors affecting thermodynamics and kinetics of G4 folding-unfolding, on structural organization of multiquadruplex systems, and on conformational features of RNA G4s and hybrid DNA-RNA G4s are discussed. Here we report the data on location of G4 sequence motifs in the genomes of eukaryotes, bacteria, and viruses, characterize G4-specific small-molecule ligands and proteins, as well as the mechanisms of their interactions with quadruplexes. New information on the structure and stability of G4s in telomeric DNA and oncogene promoters is discussed as well as proof being provided on the occurrence of G-quadruplexes in cells. Prominence is given to novel experimental techniques (single molecule manipulations, optical and magnetic tweezers, original chemical approaches, G4 detection in situ, in-cell NMR spectroscopy) that facilitate breakthroughs in the investigation of the structure and functions of G-quadruplexes.

Parallel G-Quadruplexes Formed by Guanine-Rich Microsatellite Repeats Inhibit Human Topoisomerase I.

Using UV and CD spectroscopy, we studied the thermodynamic stability and folding topology of G-quadruplexes (G4), formed by G-rich fragments in human microsatellites that differ in the number of guanosines within the repeating unit. The oligonucleotides d(GGGT)4 and d(GGT)4 were shown to form propeller-type parallel-stranded intramolecular G-quadruplexes. The G4 melting temperature is dramatically decreased (by more than 45°C) in the transition from the tri-G-tetrad to the bi-G-tetrad structure. d(GT)n-repeats do not form perfect G-quadruplexes (one-G-tetrad); folded G4-like conformation is not stable at room temperature and is not stabilized by monovalent metal ions. The minimum concentration of K+ that promotes quadruplex folding of d(GGT)4 was found to depend on the supporting Na+ concentration. It was demonstrated for the first time that the complementary regions flanking G4-motifs (as in d(CACTGG-CC-(GGGT)4-TA-CCAGTG)) cannot form a double helix in the case of a parallel G4 due to the steric remoteness, but instead destabilize the structure. Additionally, we investigated the effect of the described oligonucleotides on the activity of topoisomerase I, one of the key cell enzymes, with a focus on the relationship between the stability of the formed quadruplexes and the inhibition degree of the enzyme. The most active inhibitor with IC50 = 0.08 µM was the oligonucleotide d(CACTGG-CC-(GGGT)4-TA-CCAGTG), whose flanking G4-motif sequences reduced the extreme stability of G-quadruplex formed by d(GGGT)4.

Antitumor effect of non-steroid glucocorticoid receptor ligand CpdA on leukemia cell lines CEM and K562.

Glucocorticoids (GCs) are widely used in chemotherapy of hematological malignancies, particularly leukemia. Their effect is mediated by glucocorticoid receptor (GR), a well-known transcription factor. Besides their therapeutic impact, GCs may cause a number of side effects leading to various metabolic complications. The goal of immediate interest is testing glucocorticoid analogs capable of induction/enhancement of GR transrepression, but preventing GR dimerization and transactivation leading to side effects. In this work we have investigated effects of a promising new selective GR agonist, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride (CpdA), on CEM and K562 leukemia cells. Both cell lines express functional GR. CpdA compared with the glucocorticoid fluocinolone acetonide (FA) exerted more prominent cytostatic and apoptotic effects on the cells. Both cell lines exhibited sensitivity to CpdA, demonstrating a good correlation with the effects of FA on cell growth and viability. In contrast to FA, CpdA did not induce GR transactivation evaluated by no obvious increase in expression of GR target (and dependent) gene FKBP51. At the same time, luciferase assay showed that CpdA efficiently activated transrepression of NF-κB and AP-1 factors. We also evaluated the effect of combined action of CpdA and the proteasome inhibitor Bortezomib. The latter induced a caspase-dependent apoptosis in both T-cell leukemia cell lines. By treatment of CEM cells with different CpdA/GC and Bortezomib doses, we have designed a protocol where CpdA shows potentiating effect on Bortezomib cytotoxic activity. Generally, the present work characterizes a novel non-steroid GR ligand, CpdA, as a promising compound for possible application in leukemia chemotherapy.

Rapid photometric detection of thymine residues partially flipped out of double helix as a method for direct scanning of point mutations and apurinic DNA sites.

A spectroscopic assay for detection of extrahelical thymine residues in DNA heteroduplexes under their modification by potassium permanganate has been developed. The assay is based on increase in absorbance at 420 nm due to accumulation of thymidine oxidation intermediates and soluble manganese dioxide. The analysis was carried out using a set of 19-bp DNA duplexes containing unpaired thymidines opposite tetrahydrofuranyl derivatives mimicking a widespread DNA damage (apurinic (AP) sites) and a library of 50-bp DNA duplexes containing all types of base mismatches in different surroundings. The relation between the selectivity of unpaired T oxidation and the thermal stability of DNA double helix was investigated. The method described here was shown to discriminate between DNA duplexes with one or two AP sites and to reveal thymine-containing mismatches and all noncanonical base pairs in AT-surroundings. Comparative results of CCM analysis and the rapid photometric assay for mismatch detection are demonstrated for the first time in the same model system. The chemical reactivity of target thymines was shown to correlate with local disturbance of double helix at the mismatch site. As the spectroscopic assay does not require the DNA cleavage reaction and gel electrophoresis, it can be easily automated and used for primary screening of somatic mutations.

Genetic polymorphism and variability of chemical carcinogenesis.

Risk assessment in chemical carcinogenesis involves ratios of several factors. Individual responses of an organism to carcinogenic agents depend on polymorphism of enzymes responsible for metabolic activation/detoxification of carcinogens, DNA repair, and apoptosis, as well as promotion and progression in malignantly transformed cells. The effects of a particular polymorphic variant are manifested only in the case of its high penetrance. An integral effect is formed by the ratio of procarcinogenic and anticarcinogenic effects. The complexity of risk assessment depends on the gene polymorphism mosaic involved, directly or indirectly, in tumorigenesis and upstream/downstream interactions of gene products.

Interaction of linear homologous DNA duplexes via Holliday junction formation.

Interaction of linear homologous DNA duplexes by formation of Holliday junctions was revealed by electrophoresis and confirmed by electron microscopy. The phenomenon was demonstrated using a model of five purified PCR products of different size and sequence. The double-stranded structure of interacting DNA fragments was confirmed using several consecutive purifications, S1-nuclease analysis, and electron microscopy. Formation of Holliday junctions depends on DNA concentration. A thermodynamic equilibrium between duplexes and Holliday junctions was shown. We propose that homologous duplex interaction is initiated by nucleation of several dissociated terminal base pairs of two fragments. This process is followed by branch migration creating a population of Holliday junctions with the branch point at different sites. Finally, Holliday junctions are resolved via branch migration to new or previously existing duplexes. The phenomenon is a new property of DNA. This type of DNA-DNA interaction may contribute to the process of Holliday junction formation in vivo controlled by DNA conformation and DNA-protein interactions. It is of practical significance for optimization of different PCR-based methods of gene analysis, especially those involving heteroduplex formation.

Holliday junctions are formed in concentrated solutions of purified products of DNA amplification.

Previously, using concentrated solutions of PCR products of five different genes, we described the appearance in these solutions of DNA structures with molecular weights approximately twice greater than that of double-strand (ds) fragments and with even higher molecular weight. Since this phenomenon was shown to be not dependent on the size or sequence of the DNA fragments, we suggested that it is due to interaction of DNA duplexes. The double-sized dsDNA complex containing four polynucleotide strands of two DNA fragments was named a "tetramer". Our present work is devoted to elucidation of peculiarities of tetramer formation and its structure in solutions of a purified PCR product of p53 cDNA. We found that the intensity of tetramer formation depends on the concentration of the PCR product in solution. Three subsequent purifications of the PCR product were performed using DNA-binding matrix, but the tetramers appeared again after every procedure. After purification of PCR product preliminarily treated with S1-nuclease, tetramers appeared again, indicating that these structures are formed from dsDNA fragments. Purification of the tetramers on DNA-binding matrix led to the appearance of the initial dsDNA fragments as the main DNA structure. When electroelution and column filtration by centrifugation were used, the purification procedure was speeded up, and a solution with a higher amount of the tetramer was obtained. Electron microscopy revealed the presence of four-stranded symmetrical structures with crossing chains known as Holliday junctions. Thus, for the first time the ability of homologous dsDNA fragments to interact with the formation of Holliday junctions without participation of cell proteins has been demonstrated.