Design, synthesis and biological evaluation of 5-amino-4-(1H-benzoimidazol-2-yl)-phenyl-1,2-dihydro-pyrrol-3-ones as inhibitors of protein kinase FGFR1.

Fibroblast growth factor receptor 1 (FGFR1) plays an important role in tumorigenesis and is therefore an attractive target for anticancer therapy. Using molecular docking approach we have identified inhibitor of FGFR1 belonging to 5-amino-4-(1H-benzoimidazol-2-yl)-phenyl-1,2-dihydro-pyrrol-3-ones with IC50 value of 3.5 µM. A series of derivatives of this chemical scaffold has been synthesized and evaluated for inhibition of FGFR1 kinase activity. It was revealed that the most promising compounds 5-amino-1-(3-hydroxy-phenyl)-4-(6-methyl-1H-benzoimidazol-2-yl)-1,2-dihydro-pyrrol-3-one and 5-amino-4-(1H-benzoimidazol-2-yl)-1-(3-hydroxy-phenyl)-1,2-dihydro-pyrrol-3-one inhibit FGFR1 with IC50 values of 0.63 and 0.32 µM, respectively, and posses antiproliferative activity against KG1 myeloma cell line with IC50 values of 5.6 and 9.3 µM. Structure-activity relationships have been studied and binding mode of this chemical class has been proposed.

Chromosomal DNA balance in human stem cell line 4BL.

In the previous cytogenetic study of new human stem cell line 4BL at the 205th passage we observed the ploidy of chromosomal set and regular aberrations. To investigate the nature of monosomy of certain chromosomes the array CGH and FISH analyses have been used. The aberrations of chromosomes have been identified in all the cases of monosomies previously revealed by G-banding. The largest changes of the DNA balance have been detected in the chromosomes 2, 4, 10, 13 and 17. The probable cause of the monosomies of chromosomes 4, 10, 13 and 17 is massive loss of the genetic material. The monosomy of the second chromosome pair is caused by significant transformation one of the homologs in a type of numerous duplications and formation of der(2)t(2;?)(q21;?). Due to application of array CGH the regions of the structural aberrations of the chromosomes 2, 4, 10, 13 and 17 have been concretized, what permitted to perform their clarifying identification by multicolored FISH method. The results obtained by us confirm the hypothesis about coordinated appearance of the deletions and duplications and their stabilization impact on the transformed chromosomes.

Isolectins of phytohemagglutinin are able to induce apoptosis in HEp-2 carcinoma cells in vitro.

AIM: To study the effects of total phytohemagglutinin (PHA) and its isolectins on cell death and apoptosis in human HEp-2 carcinoma cells and to analyze the possible molecular mechanisms of lectin induced apoptosis. MATERIALS AND METHODS: The commercial preparation of the kidney beans (Phaseolus vulgaris) lectins and HEp-2 cells were used. Apoptosis index was determined using acridine orange and ethidium bromide staining. The expression levels of apoptosis mediator cleaved caspase-3 and proapoptotic Bax protein were studied by Western blot analysis. The gene expression levels were analyzed by qPCR. RESULTS: PHA and its isolectins induced apoptosis in HEp-2 cells accompanied by the increased expression of caspase-3 cleaved form, with PHA-E being the most effective. The treatment of HEp-2 cells with PHA or its isolectins resulted in a marked increase of Bax on both mRNA and protein levels. CONCLUSIONS: PHA and its isolectins were shown to induce the apoptosis in human HEp-2 carcinoma cells via increasing proapoptotic protein Bax and activating caspases-3.

Design, synthesis and biological evaluation of N-phenylthieno[2,3-d]pyrimidin-4-amines as inhibitors of FGFR1.

Fibroblast grow factor receptor 1 (FGFR1) is an important anti-cancer target that plays crucial role in oncogenesis and oncogenic angiogenesis. The structure-activity relationship (SAR) of N-phenylthieno[2,3-d]pyrimidin-4-amines was investigated. Binding of active compounds with FGFR1 kinase was analyzed by molecular modeling studies. Selected active thieno[2,3-d]pyrimidines were tested for selectivity and antiproliferative activity. The most active compounds, 3-({6-phenylthieno[2,3-d]pyrimidin-4-yl}amino)phenol and 3-({5-phenylthieno[2,3-d]pyrimidin-4-yl}amino)phenol have IC50 0.16 and 0.18 µM, respectively. The results presented here may help to identify new thienopyrimidines with optimized cell growth inhibitory activity which may be further used as anticancer agents.

[Proapoptotic properties of total phytohemagglutinine and its individual isolectins in human cell culture 4BL].

Phytohemagglutinine (PHA) is widely investigated lectin with mitogenic properties. Recently it was shown that PHA is not only cell proliferation inducer, but also has a toxic or cytostatic effect. However concentration dependence and molecular mechanisms of this effect are not enough investigated. To study proapoptotic properties of total phytohemagglutinine and its isolectins in human cell culture of not tumor origin 4BL we observed a change in the frequency of apoptotic cells in the tested cell culture under the influence of the total phytohemagglutinine and erythroagglutinin by the method of specific color luminescent dye. The activation of caspases-3 and -8 and induction of protein Bax expression under the influence of lectins were detected by Western blot analysis. It was revealed that erythroagglutinin induced apoptosis with the highest efficiency compared with leukoagglutinin and total phytohemagglutinine. The induction of apoptosis in human cell culture of not tumor origin 4BL is probably caused by activating caspase-dependent and mitochondrial signalling.

[The Wnt/beta-catenin signaling in embryonic cardiogenesis, postnatal development and myocardium reconstruction].

Wnt/beta-catenin signaling exerts great and diverse influence on the formation, development and vital activity of a great number of vertebrate tissues, including heart tissue. The role of Wnt/beta-catenin signaling and beta-catenin itself in the processes of cardiogenesis and adult myocardium functioning is not fully elucidated to date. In the current review we made the attempt to generalize data from up-to-date literature dealing with participation of this signaling pathway in embryogenesis and postnatal heart development as well as in adult myocardium functioning in normal conditions and during stress adaptation, aging, resulting in hypertrophy and heart remodeling. Based on the experimental articles and reviews we can assume that Wnt/beta-catenin signaling pathway is involved not only in controlling the cardiogenesis but in processes of adaptation and remodeling of adult organ, too. This control can be characterized as complicated and multi-step and beta-catenin appears to be a perspective candidate for new approaches development for the adult myocardium pathologies therapy.

[Comparative analysis of a new human cell line 4BL karyotype at long-term cultivation. Ploidy of chromosomal set].

Long-term cultivation of human cells, including stem cells, can lead to substantial transformation of the karyotype and occurrence of genetic instability. The aim of this research was a comparative cytogenetic study of the karyotype of a new human stem cell line 4BL at 160 and 205 passages. The absence of 10 and 13 pairs of chromosomes and the monosomy of chromosomes 4, 8, 10, 11, 13, 15, 17, 21, X were observed; also six regular marker chromosomes were detected. Chromosomes 1, 15 and 21 are involved in translocations t(l;11), t(5;15), t(12; 15), t(16;21). Modal class of the karyotype is within 41-43 chromosomes at both 160 and 205 passages. The frequency of polyploid cells have been increased from 2.8% at 160 passage up to 36% at 205 passage. Cells with a near-haploid karyotype were not detected at 205 passage (in contrast to 24.6% at 160 passages) and a decline of the level of premature separation of chromatids was observed. We assume stabilization of karyotype of the cell line 4BL at 205 passage and consider that further research is needed to predict the direction of karyotypic evolution of these cells in vitro.

[The role of some donor-host cell interactions in conditions of the regenerative process microenvironment].

The review is devoted to the analysis of experimental data about possible mechanisms of transdifferentiation or plasticity of tissue specific stem cells. Considerable attention is focused on the mechanisms and genetic consequences of fusion of different types of donor cells with the cells of recipient tissues which investigated on the models of cellular therapy of liver and heart diseases. The role of various kinds of cell contacts and their role in stem cells integration, reparation and regeneration of injured tissue and horizontal genes transfer are considered.

[The influence of lectin isoforms of Bacillus subtilis saprophytic strain IMV B-7014 on viability of normal and cancer cells in vitro].

Structural and functional polymorphism of saprophytic bacterium lectin was demonstrated to be due to subunit organization of the molecule as it was shown for many lectins of plant and animal origin. Three isoforms of extracellular sialic acid-specific lectin produced by Bacillus subtilis saprophytic strain IMV B-7014 were discovered that differed for physicochemical and biological properties. The influence of the lectin isoforms on mammalian cells proliferation and morphology in vitro depends both on the subunit organization of the protein molecule and the type of cells under study.

[Bioinformatic analysis of potential post-translational modification sites of the human O6-methylguanine-DNA methyltransferase (MGMT) protein].

Using in silico analysis a number of potential sites for post-translational modifications has been revealed within the human O6-methylguanine-DNA methyltransferase (MGMT) protein. In particular these were the acetylation of Gly3 residue in the N-terminus of protein and internal residues Lys132 and Lys135; Arg166 residue methylation; Lys63 SUMOylation and ubiquitination of Lys31, Lys39, Lys49, Lys63, Lys67, Lys135, Lys156, Lys196, Lys209. Also it has been predicted 16 novel potential phosphorylation sites of serine residues (positions 13, 124, 144, 182, 183, 190, 215, 216 and 230), tyrosine residues (positions 100 and 189) and threonine residues (positions 23, 69, 94, 126 and 229), as well as five binding sites for kinases and other proteins (Serl3 with 14-3-3, Val21 and Ile172 with D-domain, Pro78 and Pro111 with SH3-domain, Pro111 with MAPK3). Some kinases predicted by the authors are known as partners of the MGMT protein, that confirms the probability of modification of the given sites. Potential sites require further experimental confirmation of modifications and investigation of their influence on stability and DNA-repair activity of this protein.

[Influence of exogeneous proteins on mutagenic process].

Mutagenic factors of biological origin by the example of carbohydrate-binding proteins are observed. An attempt of summarizing the existing data concerning participation of exogenous macromolecules in mutagenic process is made. The mechanisms of the influence of exogenous macromolecules on mutagenesis, proliferation, and surviving of mammalian cells are discussed.

[Induction of repair enzyme O6-methylguanine-DNA methyltransferase gene expression under the influence of cytokine EMAP II in human cells in vitro].

The aim of our study was to investigate the effect of recombinant human cytokine EMAP II (endothelial monocyte-activating polypeptide II) on the expression of MGMT gene, encoding repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) in human cell cultures. The influence of EMAP II on cell proliferation was performed using routine MTT assay. Identification of MGMT in cell extracts was performed using Western blot analysis. We used cell lines: A102 (fibroblasts), CB-1 (umbilical cord blood stromal cells), 4BL6 (cells derived from peripheral blood). It was shown that cytokine EMAP II caused induction of MGMT expression in studied human cell lines. There was a decrease in cell number at high concentrations of this cytokine. It was found that the presence of cytokine EMAP II in serum-free growth medium leads to increasing of repair enzyme MGMT expression level in human cells in vitro.

[Human mobile genetic elements: structure, distribution and functional role].

Data concerning human mobile genetic elements which make up 45% of the genome are reviewed. Much attention is focused on their role in genome functioning, such as recombination, regulation of gene expression and neogenes besides classification and distribution.

[The influence of lectins on some repair processes in mammalian cells in vitro].

The influence of plant lectins on primary DNA damage repair and on expression of repair enzyme alkylguanine-DNA alkyltransferase (MGMT) in mammalian cells in vitro has been investigated. Those lectins have been shown to modify DNA damage repair process, and to induce the MGMT expression increasing its level in the used model system.

Spontaneous premature condensation of chromosomes in normal and transformed mammal cells.

AIM: To study the relation between premature chromosome condensation and the ability of the cells to undergo malignant transformation. METHODS: Standard cytogenetic analysis of bone marrow cells and cultured normal and tumor cells has been used. RESULTS: Comparative analysis of the frequency of occurrence of the cells with premature chromosome condensation (PCC) (cell "arrest" at G2/M phase) in relation to dividing cells in the cultures of human immortalized cells of hematopoietic origin, human lung carcinoma A-549 cells, and in populations of bone marrow cells of BALB/c and C57BL/6 mice differing in predisposition for myeloma development has been performed. It has been revealed that in populations of bone marrow cells of C57BL/6 mice the relation of cells with PCC to dividing ones is 2-3-fold lower than in other studied cell populations. Immortalized and malignantly transformed human cell lines were characterized by high frequency of occurrence of cells with PCC. In the cells of A-549R subline characterized by suppressed malignant phenotype this index was lower than in parental A-549 cells. CONCLUSION: The obtained data point on possible relation between disturbed passing of "check point" by cells upon transition from G2 phase of cell cycle to mitosis and increased genetic heterogeneity of new cell generation associated with ability of cells to immortalization and malignant transformation.

[Effect of lectins on induction of apoptosis in the populations of mammalian cells in vitro].

The cytotoxic action of lectins different in origin and carboxyl specificity has been studied. It has been shown that all types of lectins at high concentrations (20 mkg/ml) were able to induce apoptosis in the in vitro populations of Chinese hamster cells two days after the treatment. In the case of Persa fluviatilis lectin this effect was detected immediately after the treatment and two days later as well. It was shown that Sambucus nigra lectin did not influence the frequency of apoptosis in the culture of human cells in contrast to the Lens culinaris and P. fluviatilis lectins. The tendency of stimulation of human cell proliferation under exposure to P. fluviatilis lectin at low concentration (0.2 microg/ml) has been registered.

[Cytomorphological charasteristics of a novel mouse cell line G1].

Growth and morphological properties of a novel mouse cell line G1 have been investigated. It has been shown that cells of this cell line possess the ability of spontaneous transformation in vitro: the cells have unlimited growth in culture, grow in medium with a low serum content and form multilayer colonies on a cell monolayer and cell colonies inside agar. Using micronucleus test it has been revealed that cells of the G1 cell line possess different forms of aberrant mitosis. The results indicate the neoplastic G1 cell transformation with aberrant mitosis.

[Expression of the membrane antigens in native and cryopreserved embryonic precursor cells].

Native and cryoconservated human embryonic liver cells at different stages of gestation (5, 6 and 7 weeks) have been studied. Tissue desagregation was performed by trypsinisation technique. A part of the samples was studied after their trypsinisation, another part - after 3-week storage. Cell viability of all samples was higher than 90%. CD34 antigen expression was highest in 5-week gestation samples. The tendency of the deacrease of CD34 antigen expression was seen in 7-week gestation samples. CD34 antigen expression was higher in the samples after their cryoconservation than in native ones. The CD34 antigen expression afer their cryoconservation was accompanied by an increase in numbers of cells expressing CD38, CD45RA, CD33, CD13 and CD14 antigens as well as markers of erythroid precursor cells -CD71, CD36 and CD45RO. Percentage of alpha-actin positive cells was higher in 6- and 7-week samples. An increase in alpha-actin positive cell fraction has been seen also in samples after their cryogenic conservation.

[The possible ways of the diploidization of the polyploid germinative stem cells in the mouse BALB/c line].

Quantitative and qualitative chromosome rearrangements in the cell line G1 established from a genital ridge of the 12,5 dpc BALB/c mouse embryo were analysed. Cytogenetic analysis was performed on the 75th passage of in vitro cultivation. It has been shown that by this passage the cell population was heterogenous. It is suggested that such heterogeneity may be caused by realization of two simultaneous processes namely the cell polyploidization and their secondary diploidization. These processes were accompanied by some chromosome destructions, and the creation of small new acrocentric chromosomes and large aberrant chromosomes as well as Robertsonian translocations. The present study demonstrates in vitro karyotype evolution of the mouse cell line G1 including the increased instability of the chromosome apparatus.

[Effect of albumin on frequency of spontaneous mutations in somatic mammalian cells in vitro].

Human albumin is able to induce statistically significant increasing of the frequency of spontaneous mutations in the hprt locus in cultured somatic mammalian cells. The strait dependence of the mutagenic effect on the protein concentration has been shown. The products of albumin degradation reveal antimutagenic activity in the investigated test-system.