[Reduced Expression of the Tissue-Specific Oct-IL Isoform Exerts an Antitumor Effect on Namalwa Burkitt's Lymphoma Cells].

Increased expression levels of the Oct-1 transcription factor is considered to be one of the key markers of poor cancer prognosis. In addition to the ubiquitous Oct-1A isoform, which is found in all cells, there also exists a tissue-specific Oct-1L isoform, which is expressed in hematopoietic cells. Oct-1L increases cell resistance to different stresses and also regulates the expression of genes controlling differentiation of hematopoietic and immune system cells. The tissue-specific Oct-1L isoform levels are significantly increased in the B-cell lymphoblastoma Namalwa and Raji lines and the T-cell lymphoblastoma Jurkat line compared to normal B and T cells. Apparently, aberrant Oct-1L overexpression not only enhances stress resistance but also leads to the disruption of developmental pathways in the cells promoting their malignant transformation. We report here that targeted suppression of the tissue-specific Oct-1L isoform expression reduces the proliferation rate of Namalwa B-lymphoblastic Burkitt's lymphoma cells, significantly increases cell death rate under hypoxic conditions, and makes cells more sensitive to chemotherapeutic agents such as docetaxel and doxorubicin. These results indicate that targeted therapy aimed at the suppression of the Oct-1 isoforms with increased expression levels in tumor cells rather than the total Oct-1, thus avoiding the traumatic effects of total Oct-1 knockdown, may be promising. Selective suppression of Oct-1 isoforms is a promising strategy in the treatment of lymphoid tumors and may contribute to mitigating the disease course and increasing survival rates in cancer patients.

5-Azacytidine Suppresses the Expression of Tissue-Specific Oct-1 Isoform in Namalwa Burkitt's Lymphoma Cell Culture.

Overexpression of the transcription factor POU2F1 (Oct-1) increases the malignant potential of the tumor and determines the unfavorable prognosis for both solid and hematological cases of the disease in human carcinogenesis. The Oct-1 level determines the rate of development of the disease in acute myelodysplastic leukemia (AML), and a decrease in its expression significantly delays the development of leukemia in mice; however, a complete knockout of Oct-1 leads to the death of the animals. POU2F1 (Oct-1) is expressed as several isoforms transcribed from alternative promoters. They include both ubiquitous and tissue-specific isoforms. It was shown that in Burkitt's lymphoma Namalwa cells 5-azacytidine specifically suppresses the expression of the tissue-specific isoform Oct-1L mRNA (level of Oct-1L is abnormally increased in these cells), while not causing changes in the amount of the ubiquitous isoform Oct-1A mRNA. These results show that it is possible to selectively reduce the transcription level of the Oct-1L isoform aberrantly expressed in human tumor cells.

The Emergence of a New Isoform of POU2F1 in Primates through the Use of Egoistic Mobile Genetic Elements.

The emergence of new genes and functions is of paramount importance in the emergence of new animal species. For example, the insertion of the mobile element Tigger 2 into the sequence of the functional gene POU2F1 in primates led to the formation of a new chimeric primate-specific isoform POU2F1Z, the translation of which is activated under cellular stress. Its mRNA was found in all species of monkeys, starting with macaques. Analysis of the fragments of the Tigger2 copy corresponding to the human exon Z showed that the splicing sites of exon Z are homologous in humans and in most monkeys, with the exception of lemurs and galagos. The stop codon introduced into the mRNA by the Tigger2 sequence is present in all primates, starting with macaques. The internal ATG codon is also present in all primates, with the exception of lemurs and galagos. In the course of evolution, other MGEs, mainly of the SINE type, were inserted into the Tigger2 copy. In the course of evolution, both the location and the number of mobile SINE elements within the POU2F1 gene changed. Starting with macaques, the pattern of the arrangement of SINE elements within the Tigger2 copy in the studied region of the POU2F1 gene was fixed and then remained unchanged in other primates and humans, which may indicate its functional significance.

[Comparative Analysis of the Structure of Three piRNA Clusters in the Drosophila melanogaster Genome].

Here we attempt to reconstruct the sequence of events that led to the formation of three regulatory piRNA clusters, namely, 20A, 38C and flamenco in the Drosophila melanogaster genome. Both the 38C and flamenco clusters include inverted sequences, which potentially form double-stranded RNA hairpins. We present evidence in favor of the well-known hypothesis of piRNA clusters as "transposon traps". According to this model, the presence of the only copy of the transposon in the genome indicates that its expression is suppressed by an RNA-interference mechanism immediately after the mobile element enters the piRNA cluster. We also discuss high the structural variability of piRNAs in Drosophila clusters and cases of horizontal transmobile elements between related species.

Thapsigargin, Inhibitor of Sarco-Endoplasmic Ca2+-ATPase, Effectively Suppresses the Expression of S100A4 Protein in Human Breast Cancer Cell Line.

Thapsigargin (SERCA ATPase inhibitor) inhibited the S100A4 metastatic marker expression in MDA-MB231 breast cancer cells. We found that S100A4 gene transcription is regulated by Ca2+ signaling pathways. We found that the synthesis of S100A4 mRNA and S100A4 protein in MDA-MB231 cells was effectively suppressed by thapsigargin at a concentration of 0.4-4 µM with retaining cell viability. We assume that the change in the gene transcription in response to disturbance of Ca2+ homeostasis is directly involved in the remodeling of Ca2+ signaling pathways.

The Expression Level of S100A4 Protein Affects the Migration Activity of Breast Cancer Cells.

Reduced expression of metastatic marker protein S100A4 in triple-negative breast cancer cells MDA-MB-231 leads to a decrease in the migration ability of cells and increases the sensitivity of the modified cells to docetaxel therapy. Cells capable of migration differ from the immotile cells in the content of the S100A4 protein in the cell, and this difference persists after the treatment of cells with the agents that reduce the intracellular level of S100A4. The presence of exogenous S100A4 protein in culture medium reduces the content of this protein in breast cancer cells. The results of the study show that the ability of breast cancer cells to migrate depends on the S100A4 protein concentration in the cell.

The first complete Mag family retrotransposons discovered in Drosophila.

A retrotransposon of the Mag family was found in the Drosophila simulans genome for the first time. We also identified novel transposable elements representing the Mag family in seven Drosophila species. The high similarity between the 3' and 5' long terminal repeats in the found copies of transposable elements indicates that their retrotransposition has occurred relatively recently. Thus, the Mag family of retrotransposons is quite common for the genus Drosophila.

[Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo].

Bone morphogenetic protein-2 (rhBMP-2) represents the osteoinductive protein factor which plays a dominant role in growth and regeneration of a bone tissue. In clinical practice the bone grafting materials on the basis of rhBMP-2 are widely applied; the Russian analogues of similar materials are not produced. The fragment of the bmp2gene coding for a mature protein was cloned in Escherichia coli. The effective overproducing strain of rhBMP-2 was created on a basis of the E. coli BL21 (DE3). The rhBMP-2 production was about 25% of total cell protein. The biologically active dimeric form of rhBMP-2 was obtained by isolation and purification of protein from inclusion bodies with subsequent refolding. The rhBMP-2 sample with more than 80% of the dimeric form was obtained, which is able to interact with specific antibodies to BMP-2. Biological activity of the received rhBMP-2 samples was shown in the in vitro experiments by induction of alkaline phosphatase synthesis in C2C12 and C3H10T1/2 cell cultures. On model of the ectopic osteogenesis it was shown that received rhBMP-2 possesses biological activity in vivo, causing tissue calcification in the place of an injection. The protein activity in vivo depends on way of protein introduction and characteristics of protein sample: rhBMP-2 may be introduced in an acid or basic buffer solution, with or without the carrier. The offered method of rhBMP-2 isolation and purification results in increasing common protein yield as well as the maintenance of biologically active dimeric form in comparison with the analogues described in the literature.

[Isolation and characterization of collagen-binding domains from human von Willebrand factor].

DNA fragments encoding for two collagen binding decapeptides from the human von Willebrand factor (vWF-H1 and vWF-H2) were cloned in the Escherichia coil culture. Overproducing strains of the chimeric proteins vWF(H1)-CBD and vWF(H2)-CBD consisting of the corresponding decapeptide, Gly-Ser spacer and a cellulose binding domain (CBD) from Anaerocellum thermophilum were constructed. Using one-stage purification on cellulose, the highly purified samples of vWF(H1)-CBD and vWF(H2)-CBD proteins were obtained and the ability of these proteins to bind collagen was studied. These constructions are planned to be used for development of the recombinant collagen binding proteins with different biological activities, which, in its turn, will be used for development of the new generation products and materials for medicine, such as different kinds of implants, the coats, etc.

[Retrotransposon gtwin: structural analysis and distribution in Drosophila melanogaster strains].

A search for noncanonical variants of the gypsy retrotransposon (MDG4) in the genome of the Drosophila melanogaster strain G32 led to the cloning of four copies of the poorly studied 7411-bp gtwin element. Sequence analysis showed that gtwin belongs to a family of endogeneous retroviruses, which are widespread in the Drosophila genome and have recently been termed insect erantiviruses. The gtwin retrotransposon is evolutionarily closest to MDG4, as evident from a good alignment of their nucleotide sequences including ORF1 (the pol gene) and ORF3 (the env gene), as well as the amino acid sequences of their protein products. These regions showed more than 75% homology. The distribution of gtwin was studied in several strains of the genus Drosophila. While strain G32 contained more than 20 copies of the element, ten other D. melanogaster strains carried gtwin in two to six copies per genome. The gtwin element was not detected in D. hydei or D. virilis. Comparison of the cloned gtwin sequences with the gtwin sequence available from the D. melanogaster genome database showed that the two variants of the mobile element differ by the presence or absence of a stop codon in the central region of ORF3. Its absence from the gtwin copies cloned from the strain G32 may indicate an association between the functional state of ORF3 and amplification of the element.