Abstract: Cyclooxygenases are key enzymes in the arachidonic acid metabolism. Their unstable intermediate, prostaglandin H(2), is further metabolized to bioactive lipids by various downstream enzymes. In this study, utilizing short hairpin RNAs, we prepared a cell line of human cervix carcinoma with stable down-regulated cyclooxygenase-1 (COX-1) to assess the impact of COX-1 reduction on the downstream enzymes. We found a significant microsomal prostaglandin E synthase-1 (mPGES-1) suppression. In addition, mRNA expression of multidrug resistance protein 4 (MRP4, ABCC4), supposed to take part in antiviral and anticancer drug transport from cells, was up-regulated after COX-1 down-regulation. Our findings indicate that mPGES-1, believed to be coexpressed preferentially with cyclooxygenase-2, may be coupled to COX-1. ABCC4 up-regulation further supports the assumption of its involvement in prostanoid transport.

Abstract: The conductivity of DNA covalently bonded to a gold surface was studied by means of the STM technique. Various single- and double-stranded 32-nucleotide-long DNA sequences were measured under ambient conditions so as to provide a better understanding of the complex process of charge-carrier transport in natural as well as chemically modified DNA molecules. The investigations focused on the role of several features of DNA structure, namely the role of the negative charge at the backbone phosphate group and the related complex effects of counterions, and of the stacking interactions between the bases in Watson-Crick and other types of base pairs. The measurements have indicated that the best conductor is DNA in its biologically most relevant double-stranded form with Watson-Crick base pairs and charged phosphates equilibrated with counterions and water. All the studied modifications, including DNA with non-Watson-Crick base pairs, the abasic form, and especially the form with phosphate charges eliminated by chemical modifications, lower the conductivity of natural DNA.

Abstract: ATP-binding drug efflux transporters decrease intracellular concentrations of cytotoxic drugs, causing multidrug resistance in cancer. In this study, we examined possible interactions of ABCG2 transporter with platinum cytotoxic drugs. We demonstrate here an interference of platinum drugs with enhanced green fluorescence protein (EGFP) in the cellular models, where EGFP was employed as a reporter gene. Cytotoxicity of cisplatin (CIP), carboplatin (CAP) and oxaliplatin (OXP) was significantly lowered in MDCKII cells transfected with ABCG2 transporter and EGFP reporter. The IC(50) values in MDCKII-ABCG2 were 25.7, 164 and 165 microM for CIP, CAP and OXP, respectively, whereas IC(50) for the same cytostatics in MDCKII cells were as follows: 15.4, 133 and 50.3 microM. Addition of fumitremorgin C (FTC), a potent ABCG2 inhibitor, significantly suppressed the resistance of MDCKII-ABCG2 to OXP, suggesting that OXP interacts with ABCG2. However, FTC did not change the sensitivity of the cells to CIP and CAP. We assume that EGFP rather than ABCG2 causes the diminished toxicity of the platinum cytostatics in the transfected cells. This hypothesis was confirmed in human Hep2 cells expressing EGFP: using MTT test, IC(50) of 30.0, 247 and 27.9 microM were obtained for CIP, CAP and OXP, respectively, while 12.3, 106 and 20.5 microM were observed in the parent Hep2 cells. Employing neutral red cytotoxicity assay, similar data were obtained (IC(50) 7.73, 685 and 112 microM for CIP, CAP, and OXP, respectively, in the Hep2-EGFP cells and 1.65, 79.4 and 24.5 microM in the parent Hep2 cells). Caspase-3/7 assay revealed lower susceptibility of EGFP expressing Hep2 cells to apoptosis induced by CIP when compared to the parent cell line. We therefore conclude that EGFP in transfected cells interferes with cytotoxicity of platinum drugs by hindering the drug induced apoptosis and could cause misinterpretation of results obtained in cytotoxicity studies.

Abstract: Hydroxymethyl methacrylate-based monolithic columns for separation of oligonucleotides by capillary liquid chromatography (CLC) were prepared. We optimized composition of the polymerization mixture, which contained the monomer mixture consisting of N-(hydroxymethyl) methacrylamide (HMMAA) and ethylene dimethacrylate (EDMA), and the porogenic system composed of propane-1-ol, butane-1,4-diol and alpha, alpha'-azoisobutyronitrile (AIBN) as initiator. Separations of oligonucleotides were performed in HILIC (hydrophilic-interaction) mode using 100 mM triethylamine acetate (TEAA) in acetonitrile and in water as eluents. The influence of steepness of the mobile phase gradient on separation of the oligonucleotides was evaluated as well as the reproducibility of HMMAA monolith preparation.

Abstract: The retention behavior of the unmodified phosphodiester oligonucleotide sequence isomers was investigated on two different anion exchange columns: Biospher GMB 1000Q (based on DEAE-modified glycidyl methacrylate) and PolyWAX LP (based on silica with a crosslinked coating of linear polyethyleneimine). There was a notable difference in retention of oligonucleotides of the same composition but differing in the position of a single base. The most pronounced difference was observed between the oligonucleotides with the variable base in the end and in the center of the sequence. The use of either acetonitrile or 2-propanol as a mobile phase organic modifier did not markedly affect the retention time patterns. Prediction of the retention times of oligonucleotides must take into account the base position as well as identity. This is the first report of such a "same composition different sequence" effect, described for the short peptides, for synthetic oligonucleotides.

Abstract: The retention behavior of unmodified phosphodiester mixed-sequence oligonucleotides has been investigated under different conditions on a glycidyl methacrylate-based, DEAE-modified anion-exchange Biospher GMB 1000Q column. It was verified that separation is based not only on an anion-exchange mechanism but also on secondary interactions of the bases with the stationary phase. Notably different retention was observed for oligonucleotides differing only in the position of one base in the sequence. The nature of these secondary interactions and the effect of sequence on retention is discussed. The effect on retention of the type and concentration of organic mobile phase modifier, mobile phase pH, sample molecular weight, and the bases in the sample was investigated. A linear relationship was found between retention and the molecular weight of mixed-sequence oligonucleotides with lengths from 15 to 40. Resolution of N � 1 synthetic by-products from the full-length product was also evaluated.

Abstract: Antisense and antigene oligonucleotides (ONs) are attractive drugs for gene therapy, but major limiting factors for their routine use are inefficient cellular uptake and low accessibility to the target sites. Adding various lipophilic conjugates to the ON improves intracellular delivery as has been previously reported.We studied the cellular delivery of various ON modifications, as well as their cytosolic and nuclear distribution in mammalian Hep2-EGFP-NLS cell line. We compared uptake efficacy of ON and LNA, both conjugated with cholesterol at the 5' end. All ONs were 3' labeled with fluorescent Cy 5 dye. We made a comparison of the ONs uptake efficacy and the kinetics, both adding ONs to the culture medium, and using streptolysin-O (SL-O) permeabilization.The cellular uptake of each ON used in this study was visualized by fluorescent microscopy. We confirmed the results by FACS analysis. We determined the ratio between initial ON-chol concentration (0.4 microM) and the final amount in nucleus.SL-O can highly improve kinetics of ON delivery; not only into the cytoplasm but also to the nucleus, the presumed site of antigene ON action. The most effective nuclear uptake was observed when ON conjugated with cholesterol (ON-chol) and SL-O was used. Nuclear distribution of ON was reached within few minutes. In contrast, ON simply added to the medium reached cytoplasm only and the process of delivery took several hours.