The Problem of Apoptotic Processes Reversibility.

Apoptosis is the best understood variant of regulated cell death, which has been considered irreversible for a long time. To date, an increasing amount of data has been accumulating indicating that key events of apoptosis, such as the externalization of phosphatidylserine, mitochondrial outer membrane permeabilization, caspase activation, DNA damage, and cytoplasmic blebbing are not irreversible and can be involved in the normal cell functioning not associated with the induction of apoptosis. Anastasis - cell recovery after induction of apoptosis - can occur following elimination of proapoptotic stimuli. This can facilitate survival of damaged or tumor cells. This review describes key processes of apoptosis, which do not necessarily lead to cell death during normal cell activity as well as anastasis. Understanding mechanisms and consequences of apoptotic processes reversibility, on the one hand, could contribute to the improvement of existing therapeutic approaches for various diseases, including malignant neoplasms, and, on the other hand, could open up new possibilities for protecting cellular elements of tissues and organs from death during treatment of degenerative pathologies.

Jasmonic Acid Induces Endoplasmic Reticulum Stress with Different Outcome in Cultured Normal and Tumor Epidermal Cells.

Plant hormones produce cytotoxic effect on human cells and can trigger the processes unrelated to cell death, e.g., biosynthetic system stress. The goal of this study was to investigate activation of the endoplasmic reticulum (ER) stress by jasmonic acid (JA) and to distinguish between the responses of cultured immortalized non-tumorigenic HaCaT cells and epidermoid carcinoma A431 cells to this plant hormone. JA was used in the concentration of 2 mM, as it suppressed cell proliferation in both cell lines. We analyzed expression of genes associated with the activation of ER stress (GRP78, ATF4, CHOP), the structure of the ER and Golgi complex, and synthetic processes in the HaCaT and A431 cell lines. JA induced expression of genes responsible for the activation of ER stress and caused hypertrophic changes in the Golgi complex in both cell lines. However, the patterns of gene expression in the HaCaT and A431 cells were different, and higher levels of involucrin synthesis were observed in A431 but not in HaCaT cells, suggesting that JA activated differentiation of the tumor A431 cells only. Therefore, JA induced ER stress in both cell lines, but the consequences of ER stress were different for the epidermal immortalized non-tumorigenic and tumor cells.

Plasticity of Human THP-1 Cell Phagocytic Activity during Macrophagic Differentiation.

Studies of the role of macrophages in phagocytosis are of great theoretical and practical importance for understanding how these cells are involved in the organism's defense response and in the development of various pathologies. Here we investigated phagocytic plasticity of THP-1 (acute monocytic human leukemia) cells at different stages (days 1, 3, and 7) of phorbol ester (PMA)-induced macrophage differentiation. Analysis of cytokine profiles showed that PMA at a concentration of 100 nM induced development of the proinflammatory macrophage population. The functional activity of macrophages was assessed on days 3 and 7 of differentiation using unlabeled latex beads and latex beads conjugated with ligands (gelatin, mannan, and IgG Fc fragment) that bind to the corresponding specific receptors. The general phagocytic activity increased significantly (1.5-2.0-fold) in the course of differentiation; phagocytosis occurred mostly through the Fc receptors, as shown previously for M1 macrophages. On day 7, the levels of phagocytosis of gelatin- and Fc-covered beads were high; however, the intensity of ingestion of mannan-conjugated beads via mannose receptors increased 2.5-3.0-fold as well, which indicated formation of cells with an alternative phenotype similar to that of M2 macrophages. Thus, the type and the plasticity of phagocytic activity at certain stages of macrophage differentiation can be associated with the formation of functionally mature morphological phenotype. This allows macrophages to exhibit their phagocytic potential in response to specific ligands. These data are of fundamental importance and can be used to develop therapeutic methods for correcting the M1/M2 macrophage ratio in an organism.

Apoptosis in Cryopreserved Eukaryotic Cells.

This review considers apoptosis mechanisms that have been revealed in cryopreserved cells and which can be controlled using different chemical agents, thereby improving the viability of cells after their return to normal conditions. The role of oxidative stress as of the most significant damaging factor is discussed, as well as the reasonability of including antioxidants into cryopreservation/thawing protocols as independent agents or in combination with other compounds.

Biogenesis of Micronuclei.

The presence of micronuclei in a cell is an indicator of DNA damage and genetic instability. In this review, mechanisms of emergence of micronuclei, their functional activity, and pathways of elimination are discussed. It is supposed that morphological and functional varieties of micronuclei as well as their degradation pathways can be determined by the chromosomal material localized inside these cell structures.

Multiwalled Carbon Nanotubules Induce Pathological Changes in the Digestive Organs of Mice.

We studied the effects of regular long-term exposure to industrial nanomaterial based on multiwalled carbon nanotubules on the digestive system of mice. Nanomaterial in a concentration of 30 mg/kg was administered with drinking water over 30 days. Tissue specimens from the small intestine and liver were studied by light and electron microscopy. Multiwalled carbon nanotubules caused multiple necrotic foci in the small intestine and mixed parenchymatous degeneration in the liver. These findings suggested that multiwalled carbon nanotubules entering the digestive tract damaged intestinal villi, presumably via mechanical damage to enterocytes. It seems that multiwalled carbon nanotubules could cause degeneration indirectly, by triggering inflammatory reactions and ROS generation.

Mechanisms of Apoptosis.

Nearly 15 types of programmed cell death (PCD) have been identified to date. Among them, apoptosis is the most common and well-studied type of PCD. In this review, we discuss different apoptotic pathways in which plasma membrane and membrane organelles, such as mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, and nucleus play the pivotal role. Data concerning caspase cascades involved in these mechanisms are described. Various apoptosis induction mechanisms are analyzed and compared. The close relations between them and the possibility of switching from one pathway to another are demonstrated. In most cases, the result of these pathways is mitochondrial membrane permeabilization and/or caspase activation. These two events are closely linked and serve as the central point of integration of the apoptotic cell death pathways.

Stages of Cell Cannibalism--Entosis--in Normal Human Keratinocyte Culture.

Entosis is a type of cell cannibalism during which one cell penetrates into another cell and usually dies inside it. Researchers mainly pay attention to initial and final stages of entosis. Besides, tumor cells in suspension are the primary object of studies. In the present study, we investigated morphological changes of both cells-participants of entosis during this process. The substrate-dependent culture of human normal keratinocytes HaCaT was chosen for the work. A combination of light microscopy and scanning electron microscopy was used to prove that one cell was completely surrounded by the plasma membrane of another cell. We investigated such "cell-in-cell" structures and described the structural and functional changes of both cells during entosis. The outer cell nucleus localization and shape were changed. Gradual degradation of the inner cell nucleus and of the junctions between the inner and the outer cells was revealed. Moreover, repeated redistribution of the outer cell membrane organelles (Golgi apparatus, lysosomes, mitochondria, and autophagosomes), rearrangement of its cytoskeleton, and change in the lysosomal, autophagosomal, and mitochondrial state in both entotic cells were observed during entosis. On the basis of these data, we divided entosis into five stages that make it possible to systematize description of this type of cell death.

[Effect of plant hormones on the components of secretory pathway in human normal and tumor cells].

Plant hormones play a key role in plant growth and differentiation. Many hormones are known as potential antitumor agents, yet others appear to affect the secretory activity and are produced by mammalian cells as pro-inflammatory cytokines. The goal of this research was to study the effect of abscisic and gibberellic acids on the secretory system of human cultured epidermoid carcinoma cells A431 and keratinocytes HaCat. Immunocytochemical and morphometric analysis demonstrated that subtoxic concentration of plant hormones induced the broadening of the ER network and increased the size of Golgi complex. Electron microscopy studies confirmed the hypertrophic changes of the Golgi apparatus, specifically, the swelling of cisternae in the trans-compartment of dictyosomes after exposure to abscisic acid, and swelling of cis- and trans-compartment of dictyosomes after exposure to abscisic acid, and swelling of cis- and trans-compartments of dictyosomes after exposure to gibberellic acid. Using of Click-iT technique allowed to detect the elevation of the total protein synthesis only in A431 cells exposed to abscisic acid. Cumulative data suggests that, under these conditions, the hypertrophy of Golgi apparatus may reflect the enhanced secretory activity of cells. In other experiments, the hypertrophy of Golgi is not related to increased protein synthesis and therefore may suggest the stress-related changes of ER and Golgi apparatus. Our results demonstrate that morphologically similar reaction of cellular organelles, such as hypertrophy of Golgi apparatus, is the result of different functional activities, and that molecular mechanisms underlying the changes induced in cells need further investigations.

[application of the analytical transmission electron microscopy techniques for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in mammalian cells].

This work represents the results of the study on applicability of the modern methods of analytical transmission electron microscopy for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in A549 cell, human lung adenocarcinoma cell line. A comparative analysis of images of the nanoparticles in the cells obtained in the bright field mode of transmission electron microscopy, under dark-field scanning transmission electron microscopy and high-angle annular dark field scanning transmission electron was performed. For identification of nanoparticles in the cells the analytical techniques, energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy, were compared when used in the mode of obtaining energy spectrum from different particles and element mapping. It was shown that the method for electron tomography is applicable to confirm that nanoparticles are localized in the sample but not coated by contamination. The possibilities and fields of utilizing different techniques for analytical transmission electron microscopy for detection, visualization and identification of nanoparticles in the biological samples are discussed.

Mitochondria are targets for the antituberculosis drug rifampicin in cultured epithelial cells.

Rifampicin is a widely used drug for antituberculosis therapy. Its target is the bacterial RNA polymerase. After entry into the human or mammalian organism, rifampicin is accumulated in cells of epithelial origin (kidneys, liver, lungs) where it induces apoptosis, necrosis, and fibrosis. The purpose of this study was to determine the intracellular mechanisms leading to rifampicin-induced pathological changes and cell death. We analyzed the survival and state of the chondriome of cultured epithelial cells of the SPEV line under the influence of rifampicin. Our data show that the drug induces pronounced pathological changes in the network and ultrastructure of mitochondria, and their dysfunction results in excessive production of reactive oxygen species and release of cytochrome c. These data suggest the initiation of the mitochondrial pathway of apoptosis. Simultaneously, we observed inhibition of cell proliferation and changes in morphology of the epithelial cells toward fibroblast-like appearance, which could indicate induction of epithelial-mesenchymal transition. Thus, mitochondria are the main potential target for rifampicin in cells of epithelial origin. We suggest that similar mechanisms of pathological changes can be induced in vivo in organs and tissues accumulating rifampicin during chemotherapy of bacterial infectious diseases.

[The effect of hydroxyurea on the ciliogenesis in ciliated epithelium of mollusk Lymnaea stagnalis].

An active proliferation of the cells of ciliary epithelium in the foot of mollusk Lymnaea stagnalis was shown using radioautography. Cells labeled with 3H-tymidine were clustered into small groups. Hydroxyurea treatment decreased the proliferation of the epithelial cells. Scanning electron microscopy showed that surface of the mollusk foot was covered by extensive ciliated folds. The clusters of the cells covered with microvilli and with short cilia were localized at the bases of these folds. The cells covered with microvilli and with short cilia disappeared after 24 h of hydroxyurea treatment, ciliary epithelium appeared homogeneous and was composed of only multi-ciliated cells. Transmission electron microscopy showed no effect of the hydroxyurea on the centriole- and ciliogenesis in the multi-ciliated cells. We suggest that hydroxyurea inhibits cell proliferation and induces the differentiation of cells covered with microvilli in multi-ciliated cells.

[Selective effects of pulmonary surfactant on various subpopulations of alveolar macrophages in the model of experimental tuberculosis].

Pulmonary surfactant is necessary component for maintenance of high level of phagocytic activity of alveolar macrophages. Tuberculosis inflammation reduces the production of surfactant by type II cells and phagocytic activity of alveolar macrophages. The effects of exogenous pulmonary surfactant on the ultrastructural changes of various subpopulations of alveolar macrophages were studied by TEM-method. For investigations the bronchial alveolar lavage fluid from guinea pigs infected of M. tuberculosis and treated by isoniatid or isoniazid + exogenous pulmonary surfactant were used. It was shown that isoniazid + exogenous pulmonary surfactant normalizes the heterogeneous population of alveolar macrophages providing stimulating effects on their maturation and phagocytic activity more effectively than isoniazid therapy.

Effects of titanium dioxide nanoparticles on small intestinal mucosa in rats.

Penetration of titanium dioxide nanoparticles into enterocytes after their administration into isolated loop of rat small intestine was shown in vivo by transmission electron microscopy. Using electron diffraction, titanium dioxide nanoparticles were identified in the apical regions of the cells under plasma membranes and in deeper parts of the cytoplasm as solitary objects or small aggregations. Water dispersions of nanoparticles (3-h exposure to high concentrations) caused no appreciable morphological changes in enterocyte ultrastructure. A 28-day subacute intragastric administration of water dispersion of nanoparticles to rats led to titanium accumulation in the liver, their level was significantly higher than in the control group, which was shown by mass spectrometry with inductive-bound plasma. These data indicated the possibility of penetration of titanium dioxide nanoparticles through the gastrointestinal barrier under near-physiological conditions.

Multi-walled Сarbon Nanotubes Penetrate into Plant Cells and Affect the Growth of Onobrychis arenaria Seedlings.

Engineered nanoparticles (ENPs) are now being used in many sectors of industry; however, the impact of ENPs on the environment still requires further study, since their use, recycling, and accidental spill can result in the accumulation of nanoparticles in the atmosphere, soil, and water. Plants are an integral part of ecosystems; hence their interaction with ENPs is inevitable. It is important to understand the consequences of this interaction and assess its potential effects. The present research is focused on studying the effects of the industrial material Taunit, containing multi-walled carbon nanotubes (MWNTs), on plants, and testing of its ability to penetrate into plant cells and tissues. Taunit has been found to stimulate the growth of roots and stems and cause an increase in peroxidase activity inOnobrychis arenariaseedlings. Peroxidase activity increases with decreasing concentration of Taunit from 1,000 to 100 mg/l. MWNTs from Taunit were detected in the cells and tissues of seedling roots and leaves, implying the ability of MWNTs to penetrate into roots and accumulate there, as well as their ability to be transported into seedling leaves. Thus, the changes in the physiological parameters of plants are associated not only with MWNT adsorption on the root surface, as previously believed, but also with their penetration, uptake and accumulation in the plant cells and tissues.

[Alpha-lipoic acid triggers elimination of cells with abnormal nuclei in human carcinoma epidermoid cell line].

The skin is usually exposed to adverse environmental conditions that may cause pathological cell proliferation and cellular transformations leading to the formation of malignant cells. Antioxidants may affect these processes and induce the elimination of transformed cell. The purpose of this work was to investigate the effect of alfa-lipoic acid on human carcinoma epidermoid cell line A431. Our results showed that alfa-lipoic acid induced inhibition of cell proliferation or stimulated apoptotic cell death. Cells with abnormal nuclei were eliminated by apoptosis. Electron microscopy showed that survived cells had typical for control cells shape and organization of the nuclei, organization of the cytoplasm and organelles. Thus, alfa-lipoic acid not only triggered apoptosis of carcinoma cells, but it may also activate the mechanism of elimination of cells with abnormal chromosome number.

[Antimicrotubule agents can activate different apoptotic pathways].

The effect of agents (taxol, vincristine, and nocodazole) disturbing the microtubule network in MCF-7 human breast carcinoma cells has been examined. The aim of the study was to determine the subtypes of mitotic catastrophe and the dependence of cell death on the status of protein p53. Antimicrotubule agents can not only induce mitotic catastrophe, that is, cell death during mitosis and the death of micronucleated cells, but also activate apoptosis in interphase cells. We assume that the G1 checkpoint activation in this case occurs as a result of microtubule disruption. Apoptosis can be activated in a p53-independent manner in K-mitotic cells and after the complete disruption of the microtubule network.

[Centrosome and Golgi complex during differentiation of hepatocytes in early postnatal development of mice].

The structure and functional activity of the centrosome was analyzed in hepatocytes of 5-day old mice, as well as the lengths of Golgi complex cistemae. In the early postnatal development of mice, the liver was represented by two types of hepatocytes: in the first type hepatocytes, the centrosome was active as an organizing center of microtubules, while in the second type hepatocytes, it was inactive. It was proposed that during ontogenesis the centrosome is inactivated as an organizing center of microtubules and activated as an organizing center of intermediate filaments characteristic for differentiated hepatocytes of adult liver. Morphometry of the Golgi complex has shown that Golgi cisternae in the cell center area of early postnatal hepatocytes were longer than in the adult hepatocytes and comparable to those in G1-phase hepatocytes of regenerating liver. The possibility of relations between the differences in the Golgi complex morphology and ontogenetic changes in the functional activity of centrosomes is discussed.

[Specific features of centriole formation and ciliogenesis in ciliary epithelium cells of respiratory tracts in patients with Kartagener syndrome].

An electron microscopic study of the ciliary epithelium of respiratory tracts was carried out in children (members of the same family) with Kartagener syndrome, which is a variant of ciliary dyskinesia. It was shown that in the case of both mobile cilia and ciliary dyskinesia in man, centrioles are formed during formation of the ciliary basal bodies predominantly de novo, involving deuterosomes. A wide spectrum of pathological changes was described in literature, such as the absence of dynein arms in the axoneme and disorganization of axoneme structure. In addition to these changes in the ciliary system, we found integration of several ciliary axonemes by the same plasma membrane, running of microtubules from the plasma membrane as bundles, different orientation of basal legs, etc.