Loris Zamai

Differential kinetics of propidium iodide uptake in apoptotic and necrotic thymocytes.

Apoptosis and necrosis represent two different mechanisms by which cells die. The dynamics of cellular lesions in these two processes differ. In particular we demonstrate that plasma membrane damage, occurring as a primary event during necrosis represents, on the contrary, a delayed but massive phenomenon during apoptosis. In consequence there are different kinetics of propidium iodide incorporation by necrotic and apoptotic thymocytes. This represents the basis for the flow cytometric identification of different cellular subsets. Analysis of these subsets after sorting showed that clearly apoptotic cells, which are not able to exclude propidium iodide for long incubation periods, do not show any morphologically detectable membrane damage. The kinetics of propidium iodide incorporation in vivo in isolated rat thymocytes can therefore be used in flow cytometric analysis. This technique can be used instead of DNA staining of ethanol-treated cells or nick translation to recognize apoptotic cells, and distinguish apoptosis from necrosis, without killing the cell.

IMP dehydrogenase inhibitor, tiazofurin, induces apoptosis in K562 human erythroleukemia cells

Tiazofurin, an anticancer drug which inhibits IMP dehydrogenase, decreases cellular GTP concentration, induces differentiation and down-regulates ras and myc oncogene expression, caused apoptosis of K562 cells in a time- and dose-dependent fashion. Apoptotic cells were detected by (1) flow cytometry, (2) electron microscopy, and (3) fluorescence in situ nick translation and confocal microscopy, while the DNA ladder was not detectable. The induced apoptosis was abrogated by guanosine which replenishes GTP pools through the guanosine salvage pathways, while it was enhanced by hypoxanthine, a competitive inhibitor of GPRT. The tiazofurin-mediated apoptosis may therefore be linked with the decrease of GTP and the consequent impairment of specific signal transduction pathways. Tiazofurin induced apoptosis also in lymphoblastic MOLT-4 cells, suggesting that this action is not confined to cells of the myeloid lineage, where the differentiating effects of the drug are more pronounced.

The behaviour of nuclear domains in the course of apoptosis

Programmed cell death is activated, by different stimuli and in many cell types, to regulate cell population balance during tissue proliferation and embryogenesis. Its initial event seems to be, in most cases, the activation of a Ca2+-dependent endonuclease, causing DNA cleavage into nucleosomic fragments. Its morphological expression is characterized by deep nuclear changes, consisting of typical cap-shaped chromatin marginations, followed by nuclear fragmentation and final formation of numerous micronuclei. Cytoplasmic damage appears in a very late stage of the process and the greatest part of the phenomenon appears to take place despite good preservation of the plasma membrane and organellar component. In the present study we analyzed apoptosis in camptothecin-treated HL60 leukaemia cells, and in freshly isolated mouse thymocytes treated with dexamethasone. The process was first quantified and time monitored by flow cytometry. Subsequently the specimens were processed for morphological examination in order to investigate the behaviour of the different nuclear domains. To follow DNA and RNA localization, we utilized osmium ammine and DNase-colloidal gold cytochemical reactions. The concentration of most DNA in the cap-shaped structures was demonstrated by these reactions. Confocal microscopy of cells processed by in situ nick-translation suggested that DNA was firstly cleaved and subsequently condensed in cup-shaped structures. Despite the strong nuclear modifications, nucleoli could be clearly recognized until the late apoptotic stages.

Nuclear pores in the apoptotic cell

SummaryDuring apoptosis, nuclear pores undergo strong modifications, which are described here in five different apoptotic models. Conventional electron microscopy, supported by freeze-fracture analysis, showed a constant migration of nuclear pores towards the diffuse chromatin areas. In contrast, dense chromatin areas appear pore-free and are frequently surrounded by strongly dilated cisternae. A possible functional significance of this pore behaviour during apoptosis is discussed.

The impairment of natural killer function in the healthy aged is due to a postbinding deficient mechanism

In order to study the fine mechanisms that underlie the impairment of non-MHC-restricted cytolytic activity which occurs during human aging, we examined by multiparametric flow cytometry the binding and lytic activities of human natural killer cells. The flow analysis revealed a striking increase of the CD16+8- subset, together with a significant decrease of CD8bright cells and total T cells (CD3+). Aging had no influence on the CD8dim subset. The total lytic activity expressed by PBL as well as their binding efficiency to K562 targets were moderately but not significantly increased in the elderly. In contrast, the cytotoxicity of the single target-bound natural killer cell (i.e., lytic efficiency) was deeply impaired in aged subjects, suggesting that the NK functional impairment observed in aging is located at postbinding level.

Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry

Natural killer (NK) cells bind to K562 tumor target cells in vitro and kill them. The binding and cytotoxic activities of NK cells are tightly related to each other: degranulation of the cytotoxic effector is the basis for target cell damage and a consequence of effector-target recognition and binding. However, the two phases of NK activity, binding and killing, have always been measured separately by various methodologies and under different experimental conditions, because of the lack of a comprehensive methodology able to measure both of them at one time. Here we describe the simultaneous measurement of the binding and killing activities against K562 of resting and cytokine (IL-2 or IL-12)-stimulated NK cells by flow cytometry. NK, K562 and conjugates can be identified and measured by flow cytometry on the basis of NK mAb staining and target cells autofluorescence (Binding Plot). Within each population of the binding plot, killed targets can be identified and measured by their scatter characteristics (Cytotoxicity Plot). We show that i) the conjugate formation is enhanced in cytokine-stimulated cells, even at relatively short co-incubation times; ii) the conjugate release is also accelerated by cytokines; iii) the conjugate release is always quicker than the induction of the morphological changes in the target cell that generate its modified scattering properties.

Early events of liver regeneration in rats: a multiparametric analysis.

Abstract5-Bromodeoxyuridine (BrdU), a synthetic analogue of thymidine, has been utilized in vivo to detect the proliferation which occurs in the liver after two-thirds surgical hepatectomy. Immunocytochemical detection of BrdU incorporation has been carried out at both the morphological and flow cytometrical level, while structural changes of regenerating liver have been investigated, using Mallory-Azan-stained paraffin sections, by means of an image analyser. The results obtained show that in vivo DNA synthesis progression throughout S phase follows a pattern similar to that previously described in vitro in both 3T3 fibroblasts and Friend erythroleukemia cells and also demonstrate a precise correlation between morphological patterns of BrdU incorporating cells and their lobular distribution. Moreover, the activation of at least two proliferation waves can be detected from 18 to 34 h after hepatectomy: the former, starting from adjacent regions of contiguous lobules, apparently induces an irregular increase of lobular dimension; the latter, involving both inner and peripheral lobular domains, seems to be correlated with the appearance of nodule-like structures at the lobule periphery. In view of these results the role of the hepatic acinus and the hypothesis of a streaming of parenchymal cells during liver regeneration have been discussed.

Lineage-related susceptibility of human hemopoietic cell lines to apoptosis.

Apoptosis plays a fundamental role in shaping normal hematopoiesis. We have investigated the relationship existing between susceptibility to apoptosis and lineage commitment in hemopoietic cells.

Anti-BrdUrd labeling of newly synthesized DNA in HL-60 cells triggered to apoptosis.

Apoptosis is an active process that takes place during pre- and postnatal life. It can be viewed as the essential counterpart to cell proliferation, both phenomena being aimed at the maintenance of tissue and organ homeostasis. Because apoptosis often takes place during the S phase of the cell cycle, we describe the spatial and temporal correlation between DNA synthesis and DNA cleavage taking place in the same nucleus at the same time as a result of the action of camptothecin on proliferating HL-60 cells in vitro. The relationship between DNA synthesis and DNA fragmentation was studied at the single-cell level by bromodeoxyuridine (BrdUrd) incorporation revealed by flow cytometry, electron microscopy, and confocal microscopy. Most HL-60 cells are triggered to apoptosis during the first hour of treatment with camptothecin, and only cells in early-middle S phase are sensitive to the drug effect, whereas late S phase cells appear insensitive to camptothecin-induced apoptosis. Our data, therefore, reinforce the hypothesis of a DNA strand break threshold that may exist in the cell, beyond which the apoptotic program is activated. Moreover, DNA synthesis activity in the nucleus committed to apoptosis is gradually downregulated; after 6 h of camptothecin treatment, virtually no residual DNA replication activity can be detected in micronuclei. DNA repair does not appear to be involved in bromode-oxyuridine incorporation during the apoptotic process.

Natural killer function in flow cytometry. III: Surface marker determination of K562-conjugated lymphocytes by dual laser flow cytometry

The recognition of effector cell populations that are able to actively from conjugates with target cells is of major importance in studies of lymphocyte cytotoxicity. A number of methodologies have been described to identify the conjugates and count them, but there have been few studies of the binding capability of the different subsets of effector cells involved in the conjugation phenomenon. Here we describe a methodology that permits the study of two surface markers on lymphocytes conjugated to K562 target cells. In particular, the expression of low density CD8 (CD8dim) has been studied on both CD3+ and CD16+ lymphocytes bound to K562 target cells. Previously described methodologies, either optical microscopy or flow cytometry, were not able to identify the effector population by mAb double staining, especially in the case of antigens expressed at low density. The flow cytometric methodology described here permits the measurement of the binding activity of small lymphocyte subsets such as the CD3+ 8dim+ population. However, the method could be used to study the binding activity of any effector population defined by mAb double staining.