[Tumor-Suppressing, Immunostimulating, and Hepatotoxic Effects of Immunostimulatory RNA in Combination with Dacarbazine in a Murine Melanoma Model].

Melanoma is one of the most aggressive tumors and is accompanied by the induction of local and systemic inflammatory responses. Combinations of chemotherapeutic agents with immunotherapy are therefore commonly used for melanoma treatment. A B16 melanoma model was used to study the tumor suppressive, immunostimulating, and hepatotoxic effects of a combination of a small double-stranded immunostimulatory RNA (isRNA) with 3'-trinucleotide overhangs and the cytotoxic drug dacarbazine compared with respective monotherapies. The drugs efficiently suppressed the tumor growth and acted synergistically. Histological and immunohistochemical examinations of tumor nodes showed that the combination of isRNA and dacarbazine significantly decreased mitotic activity and more efficiently increased apoptosis in tumor tissue as compared with either monotherapy. Regardless of the treatment regimen, signs of immune activation were observed in the spleen, including an increase in the number and diameter of lymphoid follicles and the volume density of the white pulp. Destructive changes were detected in the livers of nontreated animals with B16 melanoma. Administration of isRNA in combination with dacarbazine did not cause any additional damage to liver parenchyma, while stimulating regenerative processes in hepatic tissue of tumor-bearing animals.

[Downregulation of Human Adenovirus DNA Polymerase Gene by Modified siRNAs].

Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses.

LPS-Induced Acute Lung Injury: Analysis of the Development and Suppression by the TNF-α-Targeting Aptamer.

Acute lung injury (ALI) is a specific form of lung inflammation characterized by diffuse alveolar damage, noncardiogenic pulmonary edema, as well as a pulmonary and systemic inflammation. The pathogenesis of ALI involves a cascade inflammatory response accompanied by an increase in the local and systemic levels of proinflammatory cytokines and chemokines. The development of molecular tools targeting key components of cytokine signaling appears to be a promising approach in ALI treatment. The development of lipopolysaccharide (LPS)-induced ALI, as well as the feasibility of suppressing it by an aptamer targeting the proinflammatory cytokine TNF-α, was studied in a mouse model. The TNF-α level was shown to increase significantly and remain steadily high during the development of ALI. LPS-induced morphological signs of inflammation in the respiratory system become most pronounced 24 h after induction. Intranasal administration of TNF-α-targeting aptamers conjugated with polyethylene glycol (PEG-aptTNF-α) to mice with ALI reduced the intensity of inflammatory changes in lung tissue. Assessment of the levels of potential TNF-α target genes (Usp18, Traf1, and Tnfaip3) showed that their expression levels in the lungs increase during ALI development, while declining after the application of PEG-aptTNF-α. Therefore, topical use of TNF-α- targeting aptamers may be an efficient tool for treating ALI and other inflammatory lung diseases.

Affinity modification of human chromatin with reactive derivatives of oligonucleotides.

Reaction of 4-(N-2-chloroethyl-N-methylamino)benzylphosphamides of oligonucleotides (RCl-(pT)16 and RCl-(pApC)6) with human chromatin in intact nuclei and with metaphase chromosomes has been investigated. The oligonucleotides were targeted to poly(A) and poly(TG)-repeating DNA sequences. It was found that the reagents alkylate DNA and some proteins due to specific complex formation. The affinity character of the reaction was proved by the fact that free corresponding oligonucleotides taken in excess or preliminary treatment of chromatin with S1-nuclease both prevent the biopolymers from modification. The results obtained evidence that in human chromatin there are open DNA sequences available for affinity modification with oligonucleotide derivatives. Analysis of patterns of modified proteins within these chromatin areas may give a key to the structure of these chromatin sites.

Chromatin proteins surrounding the d(G-T)n repeats and polyamine influence as revealed by photoaffinity labeling with reactive pd(A-C)6 derivatives.

The complementary-addressed modification of DNA and proteins in chromatin using photoreactive derivatives of pd(AC)6 has been studied. These oligonucleotides form complementary complexes with specific DNA sequences and modify both DNA and proteins in the vicinity of these regions, and can be used for investigation of the protein environment in DNA. We have demonstrated that photoreactive derivatives of oligonucleotides can quickly and efficiently modify chromatin proteins and seem to be promising for investigation of perturbations in chromatin structure during the cell cycle. A comparison between modified chromatin from synchronized cells has demonstrated differences in the sets of proteins modified in the S and G1/S phases of the cell cycle. An increase in spermine and spermidine concentrations leads to an increase in modification of definite chromatin proteins. It can be supposed that the B-Z transition that can be stabilized by the presence of natural polyamines is one of the reasons for the presence of single-stranded DNA regions, containing sets of (dG-dT)n and accessible for interaction with complementary oligonucleotides.

Interaction of LNA oligonucleotides with mdr1 promoter.

LNA oligonucleotides [1] can be used for targeting to double stranded DNA by the "strand invasion" mechanism. We used affinity modification by reactive oligonucleotide conjugates for investigation of oligonucleotides interaction with structured DNA. The tested LNAs and oligonucleotides of the same sequence were assayed as anti-mdr1 drugs in different cell cultures. One of the oligos, LNA79 strongly inhibited mdr1 induction in Hela cells and totally prevented activation of mdr1 in K-562.

New reagent for protein-DNA contacts footprinting.

We have found, that the reaction of o-bromobenzoic acid with Cu2+ ions can be used as a source of activated oxygen species capable of cleaving DNA. Possibility to apply this reaction for footprinting the nucleosome core in the reconstituted chromatin was demonstrated.

Expression of the MDR1 and MRP genes in patients with lymphoma with primary bone marrow involvement.

Expression of MDR1 and MRP genes in patients with low-grade and high-grade non-Hodgkin's lymphomas with primary bone marrow involvement before and after chemotherapy was investigated. The data demonstrate that overexpression of MDR1 and MRP genes in hematological malignancies elevates in patients after chemotherapy and correlates with poor clinic prognosis and more frequent recurrences of the malignancies.

Oxidative DNA cleavage by free and oligonucleotide-conjugated O-bromobenzoic acid in the presence of copper(II) ions.

o-Bromobenzoic acid was found to promote copper-dependent reactive oxygen species formation from molecular oxygen, resulting in DNA base modification and backbone cleavage. The oligonucleotide conjugate bearing 5-(4'-aminopropyl-sulfomoyl)-2-bromobenzoic acid as a reactive group was synthesized and DNA cleavage activity of this oligonucleotide conjugate was tested on a model deoxyoligonucleotide.

Silencing of c-myc expression in tumor cells by siRNA.

Suppression of c-myc protooncogene expression in KB-3-1 cells by siRNA was investigated. The siRNA duplex targeted to the exon 3 of c-myc mRNA was prepared by in vitro transcription with T7 RNA polymerase on short dsDNA-templates. It was found that incubation of KB-3-1 cells in the presence of 75 nM siRNA results in decrease of the c-myc mRNA level down to 5% of the level in the control cells and significant decline of KB-3-1 cell proliferation rate. Using 200 nM siRNA four-fold decrease of KB-3-1 cells proliferation rate was observed and this effect was stable at least 96 h after transfection.

Cu(2+)-dependent DNA-cleaving activity of arenes.

In the presence of Cu(II) ions, plasmid DNA is cleaved under physiological condition by different arenes at low concentrations. The cleavage was dependent on the presence of O2. The DNA cleavage efficiency of the designed system arene-Cu is comparable to that of the well-known DNA cleaving reagents such as phenanthroline-Cu and ascorbic acid-Cu. However in contrast to the mentioned reagents, the system arene-Cu does not require external reducing agents or H2O2.

Silencing of MDR 1 gene in cancer cells by siRNA.

Inhibition of p-glycoprotein (PGP) expression and reverse of multidrug resistance (MDR) phenotype in KB-8-5 cells by synthetic 21-bp double-stranded oligoribonucleotides were investigated. siRNA constructs for the efficient down regulation of MDR1 that are active in nanomolar concentrations and cause reversal of MDR phenotype in cells were developed.

Excessive Labeling Technique Provides a Highly Sensitive Fluorescent Probe for Real-time Monitoring of Biodegradation of Biopolymer Pharmaceuticals in vivo.

Recombinant proteins represent a large sector of the biopharma market. Determination of the main elimination pathways raises the opportunities to significantly increase their half-lives in vivo. However, evaluation of biodegradation of pharmaceutical biopolymers performed in the course of pre-clinical studies is frequently complicated. Noninvasive pharmacokinetic and biodistribution studies in living organism are possible using proteins conjugated with near-infrared dyes. In the present study we designed a highly efficient probe based on fluorescent dye self-quenching for monitoring of in vivo biodegradation of recombinant human butyrylcholinesterase. The maximum enhancement of integral fluorescence in response to degradation of an intravenously administered enzyme was observed 6 h after injection. Importantly, excessive butyrylcholinesterase labeling with fluorescent dye results in significant changes in the pharmacokinetic properties of the obtained conjugate. This fact must be taken into consideration during future pharmacokinetic studies using in vivo bioimaging.

Silencing of Her2, CCNB1 and PKC Genes by siRNA Results in Prolonged Retardation of Neuroblastoma Cell Division.

Deregulation of the expression of the genes that are involved in the control of the cell cycle impairs cellular differentiation and leads to cell death. This process can result in uncontrollable cell proliferation and, subsequently, cancer development. In this study, we examined the effect of the silencing of cancer-related genes by small interfering RNAs (siRNA) targeted at mRNAof Her2, cyclin B1 (CCNB1), and protein kinase C(PKC) on the proliferation of human cancer cells of different origins. Maximum silencing ofCCNB1,Her2(in KB-3-1, SK-N-MC, MCF-7 cells), andPKC(in MCF-7 cells) was achieved 72 h after transfection of the corresponding siRNAs, and 12 days after the transfection, the initial levels of the target mRNAs were fully recovered. Silencing ofHer2,CCNB1,andPKCdifferently effected the proliferation of the cell lines under study. The most pronounced antiproliferative action of the investigated siRNAs was observed in neuroblastoma SK-N-MC cells (3 - 10-fold reduction in the proliferation rate) even after the recovery of the initial levels of expression ofthe Her2,CCNB1, andPKС genes. The obtained data indicate that theCCNB1 andPKCgenes can be used as targets in the development of drugs for neuroblastoma treatment.

A Comparison of Target Gene Silencing using Synthetically Modified siRNA and shRNA That Express Recombinant Lentiviral Vectors.

RNA-interference is an effective natural mechanism of post-transcriptional modulation of gene expression. RNA-interference mechanism exist as in high eukaryotes both animals and plants as well in lower eukaryotes and viruses. RNA-interference is now used as a powerful tool in study of functional gene activity and many essential for fundamental biology results was obtained with this approach. Also it's widely believed that RNA-interference could be used in working out of new therapeutic medicine against malignant, infectious and hereditary diseases. One of the main problems of these developments is search of effective methods of siRNA transfer into the target cells. At present time for these purpose different sorts of transfect ions or viral transduction are used. At present article the results of comparison of inhibition of expression of oncogene AML-ET O by synthetic siRNA and by recombinant lentiviruses coding for corresponding shRNA are presented.

Sequence-specific chemical modification of chromatin DNA with reactive derivatives of oligonucleotides.

Chemical modification of the chromatin DNA with alkylating derivatives of oligothymidylate (pT)16 and oligoadenylate (pA)16 bearing 4-(N-2-chloroethyl-N-methylamino)benzylphosphamide group at the 5'-phosphate has been investigated. It was found that the derivatives do react with DNA in chromatin. The reactions occur presumably at the complementary sequences of the DNA since the reaction of the oligothymidylate derivative is inhibited by oligonucleotide (pT)16 taken in excess and is not influenced by hexadecanucleotide of a random structure. Isolated DNA does not react with the oligothymidylate derivative. It is concluded that in chromatin, DNA is partially unwound or possesses some sites which can be opened easily in the presence of complementary oligonucleotides.

Oligo(A), oligo(TG), and Alu repeats of DNA in chromatin are available for sequence-specific chemical modification with oligodeoxynucleotide derivatives.

Reaction of 4-(N-2-chloroethyl-N-methylamino) benzylphosphamides of oligonucleotides, which are targeted to the poly(A), poly(TG), and Alu repeats of eukaryotic DNA in chromatin and isolated nuclei from HeLa cells, has been investigated. It was found that the reagents alkylate DNA and some proteins due to specific complex formation. The affinity character of the reaction was proved by the fact that free corresponding oligonucleotides taken in excess or preliminary treatment of chromatin with S1 nuclease both prevent the biopolymers from the modification. Deproteinated DNA from the same cells does not react with oligonucleotide derivatives. This suggests that the chromatin DNA must have some structural features allowing oligonucleotide binding. Reactivity may be attributed to the existence of strongly negative supercoiled DNA regions containing single-stranded sequences or regions where DNA can unwind in the presence of complementary oligonucleotides. Results obtained suggest that in eukaryotic chromatin there are open DNA sequences available for affinity modification with oligonucleotide derivatives not only due to formation of triple helixes.