V. V. Vlassov

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.

Circulating DNA in the blood and its application in medical diagnosis

Circulating nucleic acids were discovered more than 30 years ago, but did not attract much attention until the past decade. This review summarizes the data on the sources of extracellular DNA circulating in the blood, features of its circulation, and pathways of its removal. The possibility of using circulating DNA in medical diagnosis is discussed.

Downregulation of activated leukemic oncogenes AML1-ETO and RUNX1(K83N) expression with RNA-interference

In the present study, we have applied the siRNA approach to reduce the expression of AML1-ETO and RUNX1(K83N) oncogenes, which are frequently found in leukemic cells. We have designed small hairpin RNAs (shRNA) for targeting AML1-ETO oncogene and a region close to the 5′-untranslated region of mRNA for the mutant RUNX1(K83N) oncogene and expressed the shRNAs in lentiviral vectors. We report a stable reduction in expression of oncogenes following the introduction of shRNAs into cells.

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.

Circulating microRNAs in lung cancer: Prospects for diagnosis, prognosis, and prediction of antitumor treatment efficacy

The review considers the main techniques to extract microRNA (miRNA) from various biological fluids (in particular, the serum and plasma), approaches to the analysis of miRNA concentration and composition, and methods to normalize the results in data analyses. Advantages and drawbacks of the methods are described. Special attention is given to circulating miRNAs, which can be used as markers for minimally invasive diagnosis, prediction of antitumor treatment efficacy, and disease prognosis in lung cancer. The review discusses the prospects and limitations that arise as the clinical significance is evaluated for miRNAs as potential tumor markers and a better understanding is gained for the roles various miRNAs play in the pathogenesis of lung cancer.

[Cholesterol-modified anti-MDR1 small interfering RNA: uptake and biological activity].

Small interfering RNAs (siRNA) are considered to be potential agents for specific gene silencing, but low the efficacy of siRNA delivery into cells limits their biomedical application. Accumulation of siRNA coupled with cholesterol residue at the 5′-end of the “sense” strand (chol-siRNA) was studied in HEK293, HepG2, SC1, and KB-8-5 cells. In the absence of a transfection agent, the levels of both carrier-free and chol-siRNAs were very low, whereas transfection agent substantially increased transfection rate in all cell lines; in HEK293, SC1, and KB-8-5 cells transfection efficiency of the chol-siRNA was higher than that of the corresponding unmodified siRNA. Biological activity of anti-MDR1-siRNAs targeted to the 557–577 nt region of the MDR1 gene mRNA was estimated as multiple drug resistance phenotype reverting activity of KB-8-5 cancer cells. The chol-siRNA induced cancer cells’ death in the presence of previously tolerated vinblastine doses more effectively than unmodified siRNA.

Mechanism of oligonucleotide hybridization with the 3′-terminal region of the yeast tRNAPhe

Interaction of yeast phenylalanine tRNA with oligonucleotides complementary to its 3′-terminal nucleotide sequence was thoroughly studied. Using the gel retardation technique, thermodynamic and kinetic parameters of the tRNA complexation in physiological conditions were determined. Analysis of dependences of the complex formation on the oligonucleotide concentration and incubation time showed that this process proceeds in two stages. At the first stage, a metastable complex of the oligonucleotide with the open, single-stranded sequence ACCA at the 3′ end of tRNA rapidly forms. The second stage involves a slow intramolecular rearrangement of the resulting metastable complex into a full-sized heteroduplex accompanied by the tRNAPhe unfolding. The data gained suggest that the RNA unfolding stage is limiting in the interaction of oligonucleotides with natural RNAs. Principles of selection of optimal hybridization probes and antisense oligonucleotides are discussed.

The enhancin gene: One of the genetic determinants of population variation in baculoviral virulence

It was established that the virulence of the North American baculovirus strain LdMNPV-45 is almost two orders of magnitude higher than the virulence of the Asian strain LdMNPV-27 and does not depend on the test host population (gypsy moth). The Asian strain carries deletions in bro-p and vef-1 genes (82 and 91%, respectively). In accordance with the published data, the product of the latter can greatly increase the virulence of the virus. This result indicates that the population polymorphism of the virulence of baculoviruses can be explained by the vef-1 gene deletion.

Modified binary hammerhead ribozymes with high catalytic activity.

A series of binary hammerhead ribozymes was designed and assessed in terms of cleavage activity and nuclease resistance. Enhanced nuclease resistance of binary ribozymes was achieved by incorporation of 2′-modified nucleotides at the selective positions along with addition of 3′-3′-linked thymidine cap. These modified binary ribozymes efficiently cleave 190-nucleotides long MDR1 mRNA fragment and display catalytic activity much higher then respective full-length analogs.

Molecular genetic markers in diagnosis of lung cancer

The review considers the main approaches to the identification of lung cancer (LC) markers, including genetic, epigenetic, protein, transcriptomic, proteomic, metabolic, and miRNA markers. Emphasis is placed on epigenetic markers, which are the most promising because epigenetic changes are among the earliest events in malignant transformation. Special attention is given to circulating tumor markers, which can be detected in easily accessible biological fluids with minimally invasive methods and may be useful for screening the risk groups for LC, diagnosing cancer before its clinical manifestation, monitoring the tumor in remission after therapy, and verifying the diagnosis based on standard clinical and instrumental methods of diagnostics. Extracellular nucleic acids (circulating in blood, circNA) are highlighted as a potential source of material for early diagnosis of LC, prediction of the efficiency of antitumor treatment, posttreatment monitoring, and disease prognosis.