Κυριακή 12 Μαΐου 2019

Cell and Tissue Biology

Multinucleated Cells Resistant to Genotoxic Factors within Human Glioblastoma Cell Lines

Abstract

Glioblastoma tumors are characterized by a high phenotype diversity among patients and by a high cellular heterogeneity within each tumor instance. Here, we report the effects of photon irradiation of various dosage and regime onto cells of two reference (T98G and A172) and two novel (T2 and R1) human glioblastoma cell lines. In contrast to the A172 and R1 lines, where all cells were perished upon relatively low-dose irradiation, a population of resistant cells within the T98G and T2 lines survived even after a high-dose irradiation. These radio-resistant cells were large single- or multinucleated non-proliferating cells, that remained viable over one month after irradiation. The cells had markedly elevated expression levels of genes coding for factors promoting growth and angiogenesis, components of the extracellular matrix, and marker genes in-herent for mesenchymal phenotype. The expression profile of these genes in the radiation-induced growth-arrested cells T98G and Т2 was similar to that of the cell lines treated with another genotoxic factor, the fotemustine. Therefore, radio-resistant growth-arrested cells are characterized by a high activity of genes encoding proteins affecting both the tumor cells and their microenvironment.



Specific Features of the Ultrastructure and Biochemical Composition of Triticum spelta L . Leaf Mesophile Cells in the Initial Period of Stress Temperature Action

Abstract

Under controlled conditions, the effect of high (40°C, 2 h) and positive low (4°C, 2 h) temperatures on the ultrastructure of mesophyll cells of the leaf and the content of photosynthetic pigments, phenols, and flavonoids in 2-week-old Triticum spelta plants was studied. The ultrastructure of the mesophyll cells of the leaf of the control plants was typical: a developed thylakoid system was clearly seen that was immersed in a fine-grained stroma in the chloroplasts of regular lenticular shape. Short-term hyperthermia caused a partial destruction of thylakoid membranes. Wave-shaped packing of grana thylakoids, significant expansion of lumen intervals, disturbance of the structural bond between the grana thylacoids and stroma thylakoids was noted. With hyperthermia, the mitochondria noticeably "swelled," while the cristae membranes became less contrasting. The number of lipid droplets increased in the cytoplasm of cells. In the leaves, the content of chlorophylls and carotenoids decreased, however, the number of common phenols and flavonoids increased. Short-term hypothermia caused intense formation of plastoglobules, and an increase in the number and size of starch grains. Destruction of thylakoid membranes was not observed. Some of the mitochondria were rounded (40%), with their size being close to the control values, and some organelles were lenticular, "dumbbell," and "cup-shaped." Under hyper- and hypothermia, the T. spelta leaf mesophyll cells showed a tendency to increase the degree of chromatin condensation in the nucleus. Under hypothermia, the content and ratio of chlorophylls and carotenoids in leaves did not differ much from the control plants, and no significant quantitative changes in the total phenols and flavonoids were recorded.



Chromosomal Instability and Evolution of Transformed Phenotype in Cell Lines Selected from Senescent Rat Embryonic Fibroblasts with Rapamycin

Abstract

The karyotype and phenotype evolution in cell lines (Rapa) obtained by rapamycin selection from rat embryonic fibroblasts (REF) has been studied. Rapamycin, an inhibitor of mTORC1 kinase, prevents replicative aging of REF cells starting after seven to ten passages in vitro. The karyotype of the established cell lines undergoes an evolution upon their cultivation in vitro. On early passages, clonal chromosome rearrangements, more specifically trisomy of chromosome 7 and translocations t(2;7) or t(4;11), were observed. Chromosome rearrangements (CR) accumulated at late passages. Simultaneously, signs of the transformed phenotype appeared: capacity for clonal growth, a decrease in contact inhibition, and high proliferation rate in the monolayer at saturating density. The evolution of the transformed phenotype was accompanied by the loss of G1/S and/or G2/M checkpoint control. Rapa lines were characterized by accumulation of γH2AX and p53BP foci, which, however, did not colocalize. Thus indicates the accumulation of unrepaired double-strand DNA breaks (DSB). The Rapa lines are shown to express the pluripotency markers Nanog and Oct3/4. The causes of genetic and karyotype instability of Rapa lines during long-term cultivation have been discussed.



Entosis and Cell Cycle in Tumor Cell Culture

Abstract

Entosis is a type of cell cannibalism during which one tumor cell invades another. The fate of the inner cell can vary. It can leave the entotic vacuole, divide within it, or be subjected to lysosome-mediated degradation. The aim of our work was to determine whether MCF7 (p53+) human breast adenocarcinoma cells and A431 (p53-) human epidermoid carcinoma cells can pass through the cell cycle during entosis. The percentage of entotic cells was 1.01 ± 0.37% in MCF7 culture and 0.42 ± 0.27% in A431 culture. It was shown that inner cells, as well as outer cells, can synthesize DNA (BrdU incorporation) and enter mitosis. Morphometric analysis showed polyploidization of outer cells. This process is most pronounced in the A431 (p53–) cell line. In addition, polyploid cells may be the preferred targets of invasion in this culture. In the MCF-7 cell line, the number of G1-phase entotic cells was higher, probably due to p53-mediated cell cycle arrest or the preferred cell invasion in the G1-phase. Overall, in tumors with active p53 protein expression, the entosis contribution to polyploidy and genetic instability of tumor cells is less than in p53 tumor cells.



Intranuclear Actin Distribution at the Four- and Eight-Cell Stages of Mouse Embryo Development

Abstract

Intranuclear distribution of actin was studied in mouse embryos in the middle and end of the four-cell stage of development, as well as in the blastomeres of uncompacted eight-cell embryos. Actin was visualized with direct fluorescence, indirect immunofluorescence and confocal microscopy. It was found that nuclei at these stages of development contained monomeric, as well as polymeric actin, differing from conventional filamentous. The pattern of actin distribution in the nuclei at the middle-stage four-cell embryos and uncompacted eight-cell embryos was similar. Comparison of this pattern with nuclear actin distribution in the two-cell embryos that we examined previously showed that at the early stages of embryo cleavage the intranuclear actin localization was not stage-specific and was stably reproduced in the nuclei of the embryos during transition from earlier to later stage of embryogenesis. Filamentous actin that had not been found in the nuclei of middle-stage four-cell embryos was detected by fluorescent phalloidin in the nuclei of the embryos at the end of a four-cell stage of development, shortly before the beginning of the third cleavage division. The appearance of nuclear filamentous actin is possibly related to the preparation of embryos to the following cleavage division.



Mononuclear Cells of Peripheral Blood in vitro. A Model of Antipsychotic Therapy Personalization

Abstract

The implementation of a unified strategy for prescribing antipsychotics has proved to be an ineffective approach to the treatment of mentally ill patients. The study of the efficiency of pharmacological drugs on models mimicking the individual pathophysiology of the patient is one means of personalized (predictive) therapy. We evaluated the mRNA level for the genes of the neurotransmitters' receptors (ADR1BHRH1HTR2АDRD1DRD2DRD4, and DRD5). These are targets of antipsychotic drugs; therefore, they can be used as the probable biomarkers of the success of the treatment of mental illnesses of schizophrenic spectrum. We used peripheral blood mononuclear cells (PBMC) in vitro as the therapeutic model. The study included 108 patients with a proved diagnosis of schizophrenia spectrum disorders, receiving a haloperidol or olanzapine as a monotherapy. The patients were divided into two groups based on their response to the pharmacotherapy (effective or ineffective). The response was estimated with psychometric analysis performed on 28 ± 2 days of the treatment. In the group with ineffective therapy, the level of expression of the studied genes in PBMC in vitro had increased with the presence of the antipsychotic drug, while, in the group of patients with positive dynamics of mental status normalization, the analyzed level of expression remained virtually unchanged. The highest significant differences for patients with different responses on the pharmacological treatment were observed for the ADR1B and HRH1 genes in the case of olanzapine therapy (Р = 0.004 and 0.038, respectively) and for the HTR2A gene in the case of haloperidol therapy (Р = 0.039). At the same time, basic levels of gene expression in non-cultivated PBMC were not associated with the patient's response to therapy. Thus, the mRNA level for genes of neurotransmission in PBMC in vitro in the presence of antipsychotics can be proposed as a biomarker for predicting the pharmacotherapy outcome for mentally ill patients.



Comparative Morphometry of Epithelial- and Fibroblast-like Type Cells in Culture

Abstract

Two morphological types, epithelial- and fibroblast-like, can be visually observed in cultivated cells. The first includes rounded and inactive cells. The cells of the second type have an elongated shape and filopodia-like outgrowths. It is obvious that these characteristics are largely subjectively determined; this complicates studying the biology of the cells adaptable and adapted to growth in culture. We have proposed a quantitative characteristic (EF indices) for their formalization. They can be used to estimate the percentage of the contribution of, respectively, filopodia (EF1), elongation or polarization (EF2), and the cell roundness (EF3) to the cell shape using the example of six different cell lines, it was demonstrated that EF1 = 15.4 ± 2.6, EF2 = 2.9 ± 1.9, and EF3 = 81.7 ± 3.2% for epithelial-like cells and 39.9 ± 5.9, 4.5 ± 0.4, and 55.6 ± 5.6% for fibroblast-like cells. It was concluded that, if the cells of two lines have differences in at least one of the indices, they can be assigned to different cell types.



Additional Prognostic Markers of Human Colon Adenocarcinoma

Abstract

The expression of potential prognostic markers of human colon adenocarcinoma in tumor and adjacent normal tissue, as well as the relationship between their expression levels was studied. The expression of potential prognostic markers (IGFBP3, AhR, CYP1A1, CYP1B1, HIF-1α, OCT4A, OCT4B, OCT4B1) was assayed with quantitative RT-PCR (qPCR). A correlation between IGFBP3 and AhR expression was found. AhR signaling was analyzed in the primary culture of human colon adenocarcinoma BSK 8 with IGFBP3 gene knockout. It was found that the enhanced expression of AhR can be an additional diagnostic criterion for colon cancer. The correlation between the expression level of HIF-1α, IGFPB3, and OCT4 isoforms and clinic stage (TNM) was not estimated. Clones with IGFBP3 gene knockout had augmented doubling time and increased resistance to the cytostatic drug 5-ftoruracile after modulated activity of dioxin receptor with its agonist—indol-3-carbinol.



CD4+ T-Cell Cycling in HIV-Infected Patients with the Discordant Immunologic Response to the Antiretroviral Therapy

Abstract

Discordant immune response to antiretroviral therapy (ART) in HIV infection is characterized by insufficient reconstitution of peripheral CD4+ T lymphocyte numbers despite suppression of virus replication. The causes of inefficient regeneration of CD4+ T cells on ART remain elusive. The paper reports the study involving two groups of HIV-positive patients: patients with discordant response to ART (n = 20) and patients with standard response to therapy (n = 21). The numbers of cycling (Ki-67+), senescent (CD57+), and exhausted (PD-1+) CD4+ T lymphocytes of varying degree of maturity have been determined in peripheral blood and lymph nodes of HIV-positive individuals. CD4+ T cell division asymmetry index after in vitro stimulation has been analyzed. Histological study of the lymph nodes of HIV-infected patients was carried out. It has been demonstrated that in the individuals with discordant immune response to ART, intensive CD4+ T cell cycling does not result in the increase in their count neither in peripheral blood, nor in the secondary lymphoid organs. At the same time, in the patients with impaired immune system recovery, increased cell proliferation is followed by the accumulation of exhausted CD4+ T lymphocytes. Moreover, patients with discordant immune response to ART revealed an asymmetric division of in vitro stimulated CD4+ T cells. The observed defects may be the cause of inefficient immune system recovery in HIV-infected patients on ART.



Analysis of NAD and NAD-Dependent Protein Deacetylation in Mouse Tissues

Abstract

Nicotinamide adenine dinucleotide (NAD) plays a key role in the vital metabolic and regulatory processes in mammals. Disturbance of the NAD level regulation is associated with the development of such serious diseases as pellagra, neurodegenerative and cardiovascular disorders, diabetes, cancer and others. This paper presents an experimental approach that allows to determine the amount of NAD+ in mouse tissues using NMR spectroscopy, as well as the level of NAD+-dependent deacetylation of proteins in the cytosol and mitochondria.



Δεν υπάρχουν σχόλια:

Δημοσίευση σχολίου