Vol 12, No 1 (2018)

Glioblastomas are tumors of neuroectodermal origin with a high degree of diversity. Tumor cells isolated from the surgical material of patients with glioblastomas are heterogeneous populations varying in morphology, phenotype, and genetic characteristics. This paper presents a description of two new glioblastoma cell lines, R1 and T2, isolated from tumor tissue of patients in 2010. We investigated their morphological and cytochemical cell characteristics and the expression of neuronal, mesenchymal, and endothelial markers, as well as the activity of genes encoding a number of growth factors, extracellular matrix proteins, and intracellular proteins typical for cells of mesenchymal origin. R1 and T2 cell lines are morphologically different. T2 cell line was characterized by the presence of multinuclear cells. In terms of the expression of β-tubulin III, MGMT, and p53 protein, R1 cell line was more heterogeneous than T2. R1 and T2 glioblastoma cell lines also differed in the presence and ratio of cell populations with mesenchymal, neuronal, and endothelial markers. Thus, neuronal markers CD133/2 and CD56 were detected only on R1 cells. Both lines were characterized by high activity of growth factor genes TGFβ1, VEGF, and FGF2(b), lower activity of EGF, and high expression of THBS1 and αSMA genes. However, the activity of most of the genes under study in R1 cells was higher than in T2 cells. The greatest difference lay in the expression of HGF, FAP, and TNC. Comparison of two new glioblastoma cell lines, R1 and T2, with the continuously cultivated lines А172 and T98G showed that R1 line had remarkable similarity with A172, while T2 glioblastoma resembled T98G cells. Apparently, the differences between R1 and T2 cell lines are determined by the properties of the initial tumors rather than the time of the cell cultivation.

Exosomes are nanosized vesicles that are secreted by many types of cells. We have found that exosomes secreted by HEK293 and HT-1080 can suppress growth and proliferation of p53-deficient cells. Upon overexpression of exogenous p53-GFP in HEK293 cells, we observed p53 protein in exosomes that were secreted by these cells. We also found endogenous p53 in exosomes that were secreted by HT-1080 cells with a higher level of p53 expression. We were able to detect endogenous p53 protein in exosomes that originated from human plasma and were transferred to p53-deficient cells. Our findings indicate that p53 protein can be transferred between cells and may play an important physiological role.

Cells isolated from a body are resistant to bacterial invasion but lose the resistance upon immortalization and transformation. In this work, interaction of immortalized 3T3 fibroblasts and their in vitro transformed analog 3T3-SV40 cells with opportunistic bacteria Serratia grimesii was studied in a conventional medium and after incubating the cells with an antioxidant N-acetylcysteine (NAC). The 3T3 cells were shown to be approximately twice less sensitive to S. grimesii infection than similar but virus-transformed 3T3- SV40 cells. Incubation of 3T3 cells with 10 and 20 mM NAC enhanced the invasion 1.6 and 2.5-fold, respectively. Under the same conditions, the invasion of 3T3-SV40 cells by the bacteria was enhanced 2.1 and 2.4- fold. These results show that 3T3 cells are more resistant to invasion by S. grimesii than 3T3-SV40 cells, and the difference is preserved after the cells are exposed to 10 and 20 mM NAC. Among the genes which expression is known to be increased by NAC, a special role plays E-cadherin shown to interact with surface proteins (invasins) of pathogenic bacteria. Incubation of 3T3 and 3T3-SV40 cells with NAC resulted in an increased expression of E-cadherin, which correlates with the increased sensitivity of these cells to invasion. Confocal fluorescence microscopy revealed, for the first time, colocalization of S. grimesii with E-cadherin of 3T3 and 3T3-SV40 cells indicating that E-cadherin can be involved in the penetration of S. grimesii into eukaryotic cells.

Diabetes Mellitus is a chronic metabolic disease marked by altered glucose homeostasis and insulin resistance. The phosphatase PTEN antagonizes the insulin-induced-PI3K-driven cascade that normally leads to GLUT4 membrane translocation. This study investigates the effect of Phenylbutyric Acid (PBA), a chemical chaperone and a potential mediator of PTEN activity, on glucose uptake in differentiated 3T3-L1 adipocytes. Adipocyte differentiation status was quantified by Oil Red O staining and the expression of AP2. Baseline and insulin-induced adipocyte glucose uptake were assayed with and without PBA treatment. Expression of GLUT1, GLUT4, PIP3, pAkt, pPTEN, and PARK-7 was examined by western blot. Plasma membrane expression of GLUT4 was determined using immunofluorescence. Leptin and adiponectin secretion was measure by enzyme-linked immunosorbent assay. PBA treatment, alone or with insulin induction, significantly increased glucose uptake in 3T3-L1 adipocytes. PBA significantly increased GLUT1 but not GLUT4 total protein expression. However, a significant increase in membrane GLUT4 protein translocation was observed. The expression of PIP3 and pAkt increased indicating enhanced PI3k pathway activity. There was a significant decrease in PTEN activity as evident by a rise in the phosphorylated form of this protein. PARK7 protein expression increased with PBA. Treating differentiated adipocytes with PBA did not alter their differentiation status, but decreased the leptin to adiponectin ratio. Conclusion: this study showed that PBA enhances adipocyte glucose uptake potentially through its effect on glucose transporter expression and/or trafficking via the PI3K signaling pathway; suggesting PBA as a possible candidate for the ancillary management of diabetes.

Actin is a permanent component of the cell nucleus involved in many nuclear processes. However, some nuclear functions of actin remain insufficiently explored. The role played by various extracellular stimuli in regulation of nuclear actin still remains enigmatic. Deviation of basic parameters of culture medium from optimal values is a member of the group of extracellular stimuli that are very important for mammalian embryos cultured in vitro. Change in culture medium pH from the level optimal for embryo homeostasis is one such signals. The purpose of this study was to investigate the intranuclear actin distribution in nuclei of two-cell mouse embryos under stress conditions induced by changes in extracellular pH. The pattern of actin localization has been tracked after short-term culturing of the embryos at optimal (pH 7.2), increased (pH 7.8), or decreased (pH 6.5) pH conditions. Analysis was carried out with confocal microscopy using methods of direct fluorescent and indirect immunofluorescent identification of actin. It has been shown that the change of culture medium pH from the optimum value is the signal that alters intranuclear actin distribution in nuclei of the embryonic cells. Culture of two-cell mouse embryos in suboptimal pH conditions (pH 6.5 and pH 7.8) induced alterations in the intranuclear actin localization, which, in particular, were expressed in accumulation of monomeric actin and the appearance of phalloidin-stainable actin in the nuclei. These changes, in our opinion, show some signs of similarity with stress-induced changes in nuclear-actin distribution, which, as has been reported earlier by a number of researchers, have been observed in the nuclei of somatic cells.

It has been proposed to use trichrome staining of histological sections for the detection of connective tissue fiber and sites for amyloid localization, as well as for increasing color contrast. After incubation in acidin–pepsin solution, sections are dewaxed and successively stained with picrofuchsin according to van Gieson, together with nuclei counterstain with hematoxylin, Congo red, and picroindigocarmine. As a result, the amyloid bound with collagen fibers was stained brick-red, collagen and reticular fibers not bound with amyloid was stained blue-green, and cytoplasm of cells not containing amyloid was stained yellow. Trichrome staining of organs affected by amyloidosis is more informative for the analysis of organs than Congo red stain.