Luigi Abbro

Cell shape and plasma membrane alterations after static magnetic fields exposure

The biological effects of static magnetic fields (MFs) with intensity of 6 mT were investigated in lymphocytes and U937 cells in the presence or absence of apoptosis-inducing drugs by transmission (TEM) and scanning (SEM) electron microscopy. Lectin cytochemistry of ConA-FITC conjugates was used to analyze plasma membrane structural modifications. Static MFs modified cell shape, plasma membrane and increased the level of intracellular [Ca++] which plays an antiapoptotic role in both cell types. Modifications induced by the exposure to static MFs were irrespective of the presence or absence of apoptotic drugs or the cell type. Abundant lamellar-shaped microvilli were observed upon 24 hrs of continuous exposure to static MFs in contrast to the normally rough surface of U937 cells having numerous short microvilli. Conversely, lymphocytes lost their round shape and became irregularly elongated; lamellar shaped microvilli were found when cells were simultaneously exposed to static MFs and apoptosis-inducing drugs. In our experiments, static MFs reduced the smoothness of the cell surface and partially impeded changes in distribution of cell surface glycans, both features being typical of apoptotic cells. Cell shape and plasma membrane structure modifications upon static MFs exposure were time-dependent. Lamellar microvilli were clearly observed before the distortion of cell shape, which was found at long times of exposure. MFs exposure promoted the rearrangement of F-actin filaments which, in turn, could be responsible for the cell surface modifications. Here we report data that support biological effects of static MFs on U937 cells and human lymphocytes. However, the involvement of these modifications in the onset of diseases needs to be further elucidated.

Differentiation of monocytic U937 cells under static magnetic field exposure.

We present here a morphological, cytochemical and biochemical study of the macrophagic differentiation of human pro-monocytic U937 cells exposed to moderate intensity (6 mT) static magnetic fields (MF). It was found that the following substances induced differentiation in U937 cells to a progressively lower degree: 50 ng/mL 12-0-tetradecanoyl-13-phorbol acetate (TPA), low concentration of glutamine (0,05 mM/L), 10% dimethyl sulfoxide (DMSO) and 100 mM/L Zn++. Differentiated U937 cells shift from a round shape to a macrophage-like morphology, from suspension to adhesion growth and acquire phagocytotic activity, the cytoskeleton adapting accordingly. Exposure to static MF at 6 mT of intensity decreases the degree of differentiation for all differentiating molecules with a consequent fall in cell adhesion and increased polarization of pseudopodia and cytoplasmic protrusions. Differentiation alone, or in combination with exposure to static MFs, affects the distribution and quantity of cell surface sugar residues, the surface expression of markers of macrophage differentiation, and phagocytotic capability. Our results indicate that moderate-intensity static MFs exert a considerable effect on the process of macrophage differentiation of pro-monocytic U937 cells and suggest the need for further studies to investigate the in vivo possible harmful consequences of this.

Kupffer cells promote lead nitrate-induced hepatocyte apoptosis via oxidative stress

BackgroundApoptosis and its modulation are crucial factors for the maintenance of liver health, allowing hepatocytes to die without provoking a potential harmful inflammatory response through a tightly controlled and regulated process. Since Kupffer cells play a key role in the maintenance of liver function, the aim of this study was to verify whether Kupffer cells are involved in the induction of liver apoptosis after i.v. injection of Pb(NO3)2 likely by secretion mechanisms.ResultsThe in vivo hepatic apoptosis, induced by Pb(NO3)2 was prevented by a pre-treatment with gadolinium chloride (GdCl3), a Kupffer cells toxicant, that suppresses Kupffer cell activity and reduces to a half the apoptotic rate. In addition, in vivo Pb(NO3)2 administration deprives hepatocytes of reduced glutathione, whereas the loss of this important oxidation-preventing agent is considerably mitigated or abolished by pre-treatment with GdCl3. However, incubation of isolated hepatocytes and Kupffer cells and HepG2 cells with Pb(NO3)2 for 24 hours induced necrotic but not apoptotic cells. Apoptosis of hepatocytes and HepG2 cells was observed only after the addition of conditioned medium obtained from Kupffer cells cultured for 24 hours with Pb(NO3)2, thus indicating the secretion of soluble mediators of apoptosis by Kupffer cells. Apoptosis in the HepG2 cells was observed upon 24-hours incubation of HepG2 cells with 1 mM buthionine sulfoximine, a glutathione depleting agent, thus showing that there is an oxidative apoptogenic pathway in HepG2 cells.ConclusionPb(NO3)2 has, at most, a direct necrotic (but not apoptogenic) effect on hepatocytes and HepG2 cells, giving a clue about the regulatory role of Kupffer cells in the induction of liver apoptosis after a single Pb(NO3)2 injection without pre-treatment with GdCl3, probably via secreting soluble factors that trigger oxidative stress in target cells.

Hepatic clearance of apoptotic lymphocytes: simply removal of waste cells?

The in situ liver recognition of apoptotic lymphocytes was studied by using different sources of lymphocytes (i.e. human, rat and mouse) and animal models (i.e. rat and mouse). Lymphocytes were induced to apoptosis using 10(-2)M cycloheximide for up to 24 hours; three types of apoptosing lymphocytes, corresponding to different stages in the apoptotic process, were described: type 1 or early apoptosis, type 2 or mature apoptosis and type 3 or late/necrotic apoptosis. When livers were in situ injected with apoptotic lymphocytes enriched for type 1 (early), 2 (mature) or 3 (late/necrotic) apoptosis, they recognized and internalized apoptosing cells, with an efficiency directly dependent on the stage of the apoptotic process. The highest recognition rate, which was, in all cases, mediated by galactose- and mannose-specific receptors, was obtained with homologous apoptotic cells (i.e. rat lymphocytes and rat liver). Moreover, the drastically reduced efficiency of recognition of human or mouse apoptotic lymphocytes when injected into rat liver, suggested the involvement also of species-specific antigens.

Lymphocytes apoptosis: young versus aged and humans versus rats.

This paper deals with a comparative study of lymphocyte apoptosis in young versus aged and humans versus rats. Apoptotic rate achieved by the use of different apoptogenic inducers, acting at different cellular levels, and cell surface modifications were analyzed. The results showed that aged human lymphocytes and freshly isolated rat lymphocytes were more prone to undergo apoptosis. Therefore, the same apoptotic signal is recognized by human and rat lymphocytes, but the extent of the answer is related to the species, to the intensity of the apoptotic stimulus and to the metabolic/developmental condition of the cells. Surface modifications (lipids and glycans), typical of apoptosis, were observed. Our data showed that cell surface changes are species and age dependent. They are early events, progressively achieved in the course of the apoptotic process involving lateral membrane movements of molecules.

Ultrastructural analysis of apoptosis induced by apoptotic U937 cells conditioned medium

Abstract Apoptosis can be induced in cycling tumor cells (i.e., U937 human monocytic cells and HepG2 cells) as well as in normal differentiated cells (i.e., human peripheral lymphocytes) by the conditioned culture medium of apoptotic U937 human monocytic cells, likely due to the release of a soluble proteic factor. The Authors described the ultrastructural effects of the conditioned culture medium on U937 human monocytic cells. Apoptosis was rapidly detected at 1 h post incubation and grew rapidly (up to 40% apoptotic cells at 4 h post incubation). The nuclei of U937 cells induced to apoptosis by the conditioned culture medium showed the canonical ultrastructural features of apoptotic nuclei, and fragmented by budding. The cytoplasms were filled with many vacuoles, mostly lipid droplets, resembling those observed in “foamy”; cells, and vesicles with intact membranes, which were recognized as swollen mitochondria: in fact, the abundant mitochondria present in healthy U937 cells were no longer detectable.

Common morphological features of apoptotic cell blebs.

Abstract A morphological study of bleb formation, which progressively and dramatically modifies the shape of U937 cells and lymphocytes induced to apoptosis, is reported. Apoptosis was induced with different compounds in both cell types. Two different kinds of blebs are described in both the apoptotic cell types: those containing remnants of nuclear chromatin and cytoplasmatic organelles, and those containing only cytoplasm. Moreover, the blebs are differentiated according to whether they contain cellular organelles or merely cytoplasm, although chromatin and organelles are rarely found in the same bleb.

Anticancer drug resistance in drug‐induced cell death of U937 cells

Abstract Cell death and subsequent post‐mortem changes are integral parts of the normal development and maturation cycle. Cell death occurs by two alternative, opposite modes: apoptosis, a programmed form of cell death, and necrosis, an unordered and accidental form. Anticancer therapies take advantage from the ability of specific drugs to induce tumour cells to die. However, drug resistance is one of the main disadvantages in cancer chemotherapy. In the present work, we investigated the effect of taxol (pa‐clitaxel), currently considered one of the most important anticancer drugs, on human monocytic leukemia U937 cells to verify drug‐induced cell death (apoptosis) or anticancer drug‐resistance. Taxol failed to induce U937 cells to apoptosis, thus showing a constitutive drug resistance of U937 cells for this substance; conversely, they were induced to a high rate of apoptosis by puromycin, cycloheximide or oxidative stress. The incubation of U937 cells with taxol simultaneously with puromycin resulted in a slight reduction of the apoptotic rate with respect to puromycin treatment alone. When cells were incubated with meta‐iodoben‐zylguanidine (MIBG), another anticancer drug, alone or in combination with taxol and puromycin, only few morphological nuclear modifications were observed compared to untreated cells. Necrosis was, for all treatments, not higher than 8%.

Nuclear changes during apoptotsis in young and aged lymphocytes from humans and rats

Abstract The early and late phases of cell death in young versus aged and in human versus rat lymphocytes were analyzed for nuclear changes. In the ultrastructural complexity of the external perinu‐clear membrane, heterochromatin, and the textural features, these were assessed in human and rat lymphocytes induced to apoptosis by different apoptotic inducers, i.e., heat shock, cycloheximide or hydrogen peroxidase, acting at different cellular levels. The same apoptotic signal was recognized by human and rat lymphocytes, but the extent of the response was related to the species, to the intensity of the apoptotic stimulus and to the metabolic/developmental condition of the cells. In particular, nuclear morphological changes, that start early during the apoptotic program and evolve progressively, are species‐ and age‐ dependent. These data suggest that in studies aimed to validate novel chemotherapeutical strategies, attention must be addressed to the choice of the experimental model.

Administration of cytoskeleton drugs during induction of apoptosis in U937 cells

Abstract The effect of administration of cytoskeleton drugs, i.e. cytochalasin B, colchicines and taxol, during the induction of apoptosis in U937 cells by means of oxidative stress (H2O2), heat shock and inhibition of protein synthesis (puromycin) was investigated. The ultrastructure of each of the “mature”; apoptotic morphologies was compared at the transmission electron microscope. Changes in the morphology of apoptotic U937 cells were found to be inducer‐de‐pendent and appeared to be influenced by the presence of cy‐toskeletal drugs. However cytoskeleton integrity was found to be a dispensable factor for the onset of apoptosis, while it was an essential one for apoptotic morphology maturation, i.e. for the formation of blebs and apoptotic bodies and for nucleus fragmentation.