V. V. Grinev

Cytotoxic effect of flavonoids on leukemia cells and normal cells of human blood.

We compared the cytotoxic effect of 11 flavonoids on chronic myeloid leukemia (erythroblast crisis) K562 cells and peripheral blood mononuclear cells from healthy donors. Baicalein and myricetin had a specific cytotoxic effect on leukemia cells.

Viral Vectors for Stable Transduction of Human Mesenchymal Stem Cells: Systems Based on Adeno-Associated Viruses and Lentiviruses

We compared the efficiency of transduction with lentivectors based on HIV-1 and adeno-associated viruses serotype 2 and stability of transgene expression in human mesenchymal bone marrow stem cells.

The determinants of alternative RNA splicing in human cells

Alternative splicing represents an important level of the regulation of gene function in eukaryotic organisms. It plays a critical role in virtually every biological process within an organism, including regulation of cell division and cell death, differentiation of tissues in the embryo and the adult organism, as well as in cellular response to diverse environmental factors. In turn, studies of the last decade have shown that alternative splicing itself is controlled by different mechanisms. Unfortunately, there is no clear understanding of how these diverse mechanisms, or determinants, regulate and constrain the set of alternative RNA species produced from any particular gene in every cell of the human body. Here, we provide a consolidated overview of alternative splicing determinants including RNA–protein interactions, epigenetic regulation via chromatin remodeling, coupling of transcription-to-alternative splicing, effect of secondary structures in pre-RNA, and function of the RNA quality control systems. We also extensively and critically discuss some mechanistic insights on coordinated inclusion/exclusion of exons during the formation of mature RNA molecules. We conclude that the final structure of RNA is pre-determined by a complex interplay between cis- and trans-acting factors. Altogether, currently available empirical data significantly expand our understanding of the functioning of the alternative splicing machinery of cells in normal and pathological conditions. On the other hand, there are still many blind spots that require further deep investigations.

RUNX1T1/MTG8/ETO gene expression status in human t(8;21)(q22;q22)-positive acute myeloid leukemia cells

The RUNX1-RUNX1T1 fusion gene, a product of the nonhomologous balanced translocation t(8;21)(q22;q22), is a complex genetic locus. We performed extensive bioinformatic analysis of transcription initiation as well as transcription termination sites in this locus and predicted a number of different RUNX1T1 transcripts. To confirm and quantify the RUNX1T1 gene expression, we analyzed samples from seven acute myeloid leukemia (AML) patients and from the Kasumi-1 cell line. We found variable activity of the four predicted RUNX1T1 promoters located downstream of the chromosome breakpoint. Nineteen alternative RUNX1T1 transcripts were identified by sequencing at least seventeen of which predictably can be translated into functional proteins. While the RUNX1T1 gene is not expressed in normal hematopoietic cells, it may participate in t(8;21)(q22;q22)-dependent leukemic transformation due to its multiple interactions in cell regulatory network particularly through synergistic or antagonistic effects in relation to activity of RUNX1-RUNX1T1 fusion gene.

Structural and thermodynamic features of intergenic and intronic human primary microRNAs

Micro RNAs are small, noncoding RNAs involved in the regulation of gene expression in eukaryotes. Being transcribed in the nucleus in the form of a long primary precursor, a primary microRNA undergoes multistep biogenesis that leads to the formation of a mature microRNA. One of the critical biogenesis steps is the recognition and processing of primary microRNAs by microprocessor complex. Secondary structure of a primary microRNA is considered to play a key role during this biogenesis step, but specific structural motives have not yet been identified. As a result of a study on structural and thermodynamic features of intergenic and intronic microRNAs, we have demonstrated that the two main classes of human microRNAs have no statistically significant difference in respect to these parameters. The conclusion may be a base for combining the two class microRNA sequences in a common set for the search for new microRNAs. Specific structural and thermodynamic features in human primary microRNA transcript hairpins have been found: the data may be used for search of new microRNA candidates in the human genome and for a design of artificial microRNA sequences for gene therapy and functional genomics purposes.

Design and Quality Control of Short Interfering RNA

This review is devoted to analyzing design rules, as well as quality controls, in the development of highly effective and specific short interfering RNA (siRNA). Four crucial steps in the development of this siRNA are discussed, i.e., selecting target RNA and designing sense and antisense strands, as well as assessing the activity and specificity of corresponding siRNA. Special consideration is given to the principles of siRNA construction based on both structural and thermodynamical features and the nucleotide composition of siRNA, as well as structural and thermodynamical properties of target RNA and features of experimental performance. Bioinformatics resources for developing siRNA are also discussed. Information from this review can be useful for the development of highly effective and specific siRNA, short hairpin RNA (shRNA), and/or artificial microRNA (amiRNA) sequences for experimental gene therapy and functional genomics.

Multidimensional control of cell structural robustness.

Ample adaptive and functional opportunities of a living cell are determined by the complexity of its structural organisation. However, such complexity gives rise to a problem of maintenance of the coherence of inner processes in macroscopic interims and in macroscopic volumes which is necessary to support the structural robustness of a cell. The solution to this problem lies in multidimensional control of the adaptive and functional changes of a cell as well as its self‐renewing processes in the context of environmental conditions. Six mechanisms (principles) form the basis of this multidimensional control: regulatory circuits with feedback loops, redundant inner diversity within a cell, multilevel distributed network organisation of a cell, molecular selection within a cell, continuous informational flows and functioning with a reserve of power. In the review we provide detailed analysis of these mechanisms, discuss their specific functions and the role of the superposition of these mechanisms in the maintenance of cell structural robustness in a wide range of environmental conditions.

Highly efficient transfer and stable expression of two genes upon lentivirus transduction of mesenchymal stem cells from human bone marrow

The efficiency of human bone marrow (BM) mesenchymal stem cell (MSC) transduction with a bicistronic lentivirus vector was estimated, and the stability of transgene expression in genetically modified MSCs was determined. First-passage BM MSCs were capable of efficient transduction with the bicistronic lentivirus vector. The transduction efficiency depended on the multiplicity of infection (MOI), being 64.64 ± 6.5 and 88.6 ± 2.9% at MOI 10 and 20, respectively. The lentivirus transduction efficiency proved independent on the number of passages of a BM MSC culture, and expression of the egfp and dsRed1 transgenes in genetically modified MSCs remained stable for one month of culturing. A comparison showed that the level of egfp and dsRed1 transgene expression was preserved upon hepatogenic differentiation in vitro. The results provide a basis for further development of multigenic modification of human BM MSCs for research and/or therapeutic purposes.

Mapping of the 3' End for Artificial Human MicroRNAs

The 3′ end was exactly mapped for has-mir-30a-like artificial human microRNAs that specifically recognize the mRNA of the aml1/eto fusion oncogene. The results indicated that the intracellular microRNA pool was heterogeneous in linear size relative to the 3′ end, which is necessary to consider in designing and using artificial microRNAs specific for mRNAs of other genes.

Different Mechanisms of Resistance to Lymphokine-activated Killer (LAK) Cells in Leukemic Cell Sublines Selected by Etoposide or by LAK Cells.

Two models of in vitro induction of human IM-9 lymphoblastoid cell line resistance to LAK cell-mediated killing were compared. IM-9 cell line variants were selected in vitro by prolonged exposure to LAK cells or to etoposide. Sensitivity to killing by LAK cells or by etoposide, conjugation with LAK cells and surface marker patterns were compared. Both LAK-cell-selected cell subline (IM-9/SL-15) and etoposide-selected cell subline (IM-9/ER) have acquired resistance to LAK cell-mediated death. IM-9/ER cells, but not IM-9/SL-15 cells, were 3.2-fold less sensitive to etoposide as compared to parental IM-9 cells. IM-9/SL-15 cells revealed decreased conjugation with LAK cells and augmented CDlla/CD18 surface molecule expression as compared to IM-9 line. In contrast, IM-9/ER cells displayed a level of conjugation with LAK cells equal to IM-9 cells, but loss of CD11a/C18 expression. Our results suggest different mechanisms of acquired resistance to LAK cells are operative in etoposide- or LAK-selected sublines, involving deterioration of LFA-1 molecule expression and altered conjugation properties, respectively.