In vitro transfection mediated by dendrigraft poly(L-lysines): the effect of structure and molecule size.

Dendritic poly(L-lysines) (DGL) constitute promising nanomaterials applicable as a nonviral gene-delivery vector. In this study, we evaluate the transfection abilities of four DGL generations with special emphasis on the systematic description of the relationship of how generation (i.e., molecule size) affects the transfection efficacy. Using Hep2 cells, we demonstrated that the capability of unmodified DGL to deliver plasmid is of a magnitude lower than that of jetPEI. On the other hand, employing the Hep2 cell line stably transduced with eGFP, we observed that DGL G5 delivers the siRNA oligonucleotide with the same efficiency as Lipofectamine 2000. In further experiments, it was shown that DGL affords excellent ability to bind DNA, protect it against DNase I attack, and internalize it into cells.

Mechanisms of ellipticine-mediated resistance in UKF-NB-4 neuroblastoma cells.

Most high-risk neuroblastomas develop resistance to cytostatics and therefore there is a need to develop new drugs. In previous studies, we found that ellipticine induces apoptosis in human neuroblastoma cells. We also investigated whether ellipticine was able to induce resistance in the UKF-NB-4 neuroblastoma line and concluded that it may be possible after long-term treatment with increasing concentrations of ellipticine. The aim of the present study was to investigate the mechanisms responsible for ellipticine resistance. To elucidate the mechanisms involved, we used the ellipticine-resistant subline UKF-NB-4(ELLI) and performed comparative genomic hybridization, multicolor and interphase FISH, expression microarray, real-time RT-PCR, flow cytometry and western blotting analysis of proteins. On the basis of our results, it appears that ellipticine resistance in neuroblastoma is caused by a combination of overexpression of Bcl-2, efflux or degradation of the drug and downregulation of topoisomerases. Other mechanisms, such as upregulation of enzymes involved in oxidative phosphorylation, cellular respiration, V-ATPases, aerobic respiration or spermine synthetase, as well as reduced growth rate, may also be involved. Some changes are expressed at the DNA level, including gains, amplifications or deletions. The present study demonstrates that resistance to ellipticine is caused by a combination of mechanisms.

Charge transport in DNA oligonucleotides with various base-pairing patterns.

We combined various experimental (scanning tunneling microscopy and Raman spectroscopy) and theoretical (density functional theory and molecular dynamics) approaches to study the relationships between the base-pairing patterns and the charge transfer properties in DNA 32-mer duplexes that may be relevant for identification and repair of defects in base pairing of the genetic DNA and for DNA use in nanotechnologies. Studied were two fully Watson-Crick (W-C)-paired duplexes, one mismatched (containing three non-W-C pairs), and three with base pairs chemically removed. The results show that the charge transport varies strongly between these duplexes. The conductivity of the mismatched duplex is considerably lower than that of the W-C-paired one despite the fact that their structural integrities and thermal stabilities are comparable. Structurally and thermally much less stable abasic duplexes have still lower conductivity but not markedly different from the mismatched duplex. All duplexes are likely to conduct by the hole mechanism, and water orbitals increase the charge transport probability.

The role of aryl hydrocarbon receptor in regulation of enzymes involved in metabolic activation of polycyclic aromatic hydrocarbons in a model of rat liver progenitor cells.

In contrast to hepatocytes, there is only limited information about the expression and activities of enzymes participating in metabolic activation of environmental mutagens, including polycyclic aromatic hydrocarbons (PAHs), in liver progenitor cells. In rat liver "stem-like" WB-F344 cell line, sharing many characteristics with rat liver progenitor cells, PAHs are efficiently activated to their ultimate genotoxic metabolites forming DNA adducts. The present study aimed to characterize expression/activities of enzymes of two major pathways involved in the metabolism of benzo[a]pyrene (BaP): cytochrome P450 (CYP) family 1 enzymes and cytosolic aldo-keto reductases (AKRs). We report here that, apart from induction of CYP1A1 and CYP1B1 expression and the corresponding enzymatic activity, both BaP and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced rat 3alpha-hydroxysteroid dehydrogenase (AKR1C9) expression and activity. In contrast, the aldehyde reductase AKR1A1 was not induced by either treatment. Thus, both CYP1 and AKR metabolic pathways were inducible in the model of liver progenitor cells. BaP and TCDD were efficient inducers of NAD(P)H:quinone oxidoreductase 1 (NQO1) expression and activity in WB-F344 cells, a principal enzyme of cellular antioxidant defense. Both compounds also induced expression of transcription factor NRF2, involved in control of enzymes protecting cells from oxidative stress. However, although BaP induced a significant formation of reactive oxygen species, it did not induce expression of heme oxygenase-1, suggesting that induction of oxidative stress by BaP was limited. Using shRNA against the aryl hydrocarbon receptor (AhR), we found that similar to CYP1A1 and CYP1B1, the AKR1C9 induction was AhR-dependent. Moreover, constitutive AKR1C9 levels in AhR-deficient rat BP8 hepatoma cells were significantly lower than in their AhR-positive 5L variant, thus supporting possible role of AhR in regulation of AKR1C9 expression. Taken together, both CYP1 and AKR1C9 appear to be AhR-regulated metabolic pathways, which may contribute to formation of pro-carcinogenic PAH metabolites in liver progenitor cells.

COX-1 is coupled with mPGES-1 and ABCC4 in human cervix cancer cells.

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.

Studying liver regeneration by means of molecular biology: how far we are in interpreting the findings?

Liver regeneration in mammals is a unique phenomenon attracting scientific interest for decades. It is a valuable model for basic biology research of cell cycle control as well as for clinically oriented studies of wide and heterogeneous group of liver diseases. This article provides a concise review of current knowledge about the liver regeneration, focusing mainly on rat partial hepatectomy model. The three main recognized phases of the regenerative response are described. The article also summarizes history of molecular biology approaches to the topic and finally comments on obstacles in interpreting the data obtained from large scale microarray-based gene expression analyses.

Effect of ABCG2 on cytotoxicity of platinum drugs: interference of EGFP.

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.

Conductivity of natural and modified DNA measured by scanning tunneling microscopy. The effect of sequence, charge and stacking.

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.

Hydroxymethyl methacrylate-based monolithic columns designed for separation of oligonucleotides in hydrophilic-interaction capillary liquid chromatography.

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.

Cholesterol conjugated oligonucleotide and LNA: a comparison of cellular and nuclear uptake by Hep2 cells enhanced by streptolysin-O.

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.