Biomedical subjects

E Falcieri

Publications and source records attributed to E Falcieri.

At least 19 recordsLinked to original sources

PubMed2002

Actin involvement in apoptotic chromatin changes of hemopoietic cells undergoing hyperthermia.

During apoptosis, cell chromatin undergoes characteristic morphological changes, which have been long described in a variety of experimental models but are mostly not yet understood. The aim of the present study was to investigate the mechanisms underlying this phenomenon and the possible role of cytoskeleton, in particular actin. The chosen apoptotic model were HL60 hemopoietic cells undergoing hyperthermia and the starting point was the observation of thin filament bundles in decondensed chromatin of their early apoptotic nuclei. The characterization of these structures was undertaken by cytochemical, fluorescent and immunogold techniques, directed to actin identification. Taken together, our results suggest, in apoptotic cells, a deep actin rearrangement. Moreover, this cytoskeletal component, never present in normal nucleus, appears in the early apoptotic one, where it can be found in polymerized form, promptly recognizable both by conventional and immunogold electron microscopy. We suggest that, similarly to the role played by nuclear matrix in interphase and mitotic nucleus, actin could be directly involved in chromatin rearrangement occurring in apoptosis.

Actins↗

PubMed2002

The controversial nuclear matrix: a balanced point of view.

The nuclear matrix is defined as the residual framework after the removal of the nuclear envelope, chromatin, and soluble components by sequential extractions. According to several investigators the nuclear matrix provides the structural basis for intranuclear order. However, the existence itself and the nature of this structure is still uncertain. Although the techniques used for the visualization of the nuclear matrix have improved over the years, it is still unclear to what extent the isolated nuclear matrix corresponds to an in vivo existing structure. Therefore, considerable skepticism continues to surround the nuclear matrix fraction as an accurate representation of the situation in living cells. Here, we summarize the experimental evidence in favor of, or against, the presence of a diffuse nucleoskeleton as a facilitating organizational nonchromatin structure of the nucleus.

Animals↗

PubMed2002

Morpho-functional changes in human tendon tissue.

Insertion tissue biopsies of right arm common extensor tendons from 11 patients with chronic lateral epicondylitis were processed for light and electron microscopy. The subjects were aged between 38 and 54 years (only one was 25). The specimens showed a variety of structural changes such as biochemical and spatial alteration of collagen, hyaline degeneration, loss of tenocytes, fibrocartilage metaplasia, calcifying processes, neovascularization and vessel wall modifications. Tissue alterations were evident in limited zones of the tendon fibrocartilage in which the surgical resection was generally visible. The areas where the degenerative processes were localized, were restricted and in spatial contiguity with morphologically normal ones. The observed cases presented histological and electron microscopic findings that characterize lateral epicondylitis as a degenerative phenomenon involving all tendon components.

Adult↗

PubMed2001

Modulation of caspase-3 activity by zinc ions and by the cell redox state.

It is known that DNA fragmentation during apoptosis is controlled by a number of factors, a crucial step being the caspase-operated cleavage of ICAD, the DNase inhibitor. We have previously demonstrated that hydrogen peroxide-treated lymphocytes undergo apoptosis without formation of a DNA ladder; however, the use of micromolar amounts of a Zn(2+) chelator allowed DNA cleavage at internucleosomal sites. Such results were extended in the present work, thus allowing their framing into the events related to alterations in the redox state of the cell. Apoptosis in hydrogen peroxide-treated lymphocytes was found to occur with caspase-3 activation, but the enzyme activity was found to be impaired, thus affecting internucleosomal fragmentation as well as nuclear morphology. Caspase-3 activity was found to resume upon mild Zn(2+) chelation. These results provide as well an experimental model from which apoptotic events upstream and downstream of caspase-3 activity can be examined.

Adult↗

PubMed2000

Ultrastructural characterization of maturation, platelet release, and senescence of human cultured megakaryocytes.

The aim of this study was to evaluate the ultrastructural features of human megakaryocytes cultured in vitro. For this purpose, pluripotent CD34(+) (cluster of differentiation 34) hematopoietic progenitor cells, obtained from the peripheral blood of healthy adult donors, were differentiated along the megakaryocytic lineage in liquid cultures by the addition of the megakaryocyte-specific growth factor thrombopoietin (TPO, 100 ng/ml). After only 6-8 days, virtually all of the CD34-derived cells expressed the early megakaryocytic CD61 antigen, while, after 15-16 days, most cells also expressed the late megakaryocytic CD42a antigen. Ultrastructural analysis of cells obtained after 7 days of culture showed aspects typical of developing megakaryocytes (MK), such as formation of platelet territories and cytoplasmic fragmentation. At later (15-16 day) culture times, two distinct cell populations were observed: fully developed megakaryocytes releasing platelets into the culture medium and senescent megakaryocytes, characterized by morphological features of apoptosis. Analysis of DNA fragmentation in these cells revealed that apoptosis in megakaryocytes occurred in the absence of the internucleosomic cleavage, which is characteristic of most, but not all, types of apoptosis in cells of hematopoietic origin. On the other hand, flow cytometry of the DNA content of senescent megakaryocytes showed a subdiploid peak that was likely due to a loss of micronuclei during processing.

Aging↗

PubMed2000

Lineage-related sensitivity to apoptosis in human tumor cells undergoing hyperthermia.

In this study the role of hyperthermia as an apoptotic trigger was analyzed in four human tumor cell lines: HL60, U937, DOHH2, and K562. These cell lines were chosen because of their well known and different expression of bcl-2 and bcr-abl genes, the expression of which is known to be an antiapoptotic condition. HL60 and U937 cells were strongly susceptible to heat exposure, while DOHH2 cells were weakly sensitive and K562 cells were resistant, thus suggesting a possible gene involvement in this type of programmed cell death. The mechanisms underlying this apoptosis were investigated by flow cytometry, agarose gel electrophoresis, and light and electron microscopy. A subdiploid peak and DNA laddering, both of which are parameters specifically correlated to programmed cell death, were present in HL60 and U937 and, even if less evident, in DOHH2 cells undergoing hyperthermic treatment, and were absent in K562 cells. In addition, DNA single-strand cleavage was revealed by in situ nick translation, observed by confocal microscopy. Morphological analysis confirmed these results and revealed the typical chromatin changes, followed by the appearance of micronuclei and apoptotic bodies.

Apoptosis↗

PubMed2000

Programmed cell death in 2',3'-dideoxycytidine-resistant human monoblastoid U937 cells.

2',3'-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O2 and nitric oxide. Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.

Anti-HIV Agents↗

PubMed2000

Matrix metalloproteinases expression in HL-60 promyelocytic leukemia cells during apoptosis.

Human promyelocytic leukemia HL-60 cells have been used as a model to study both the expression of matrix-metalloproteinases and the mechanisms of programmed cell death. In the present study we examined the expression of these proteases in HL-60 cells stimulated by different apoptotic triggers. As shown by zymography, HL-60 cells released three major isofroms of the matrix-degrading proteases; when the leukemic cells were grown in serum-free conditions, as well as after hyperthermia and methotrexate treatment, we found a significant loss of the constitutive production of the 92 kDa matrixmetalloprotease, with an unequivocable molecular and ultrastructural evidence of programmed cell death. These results suggest that in HL-60 cells the expression/release of matrix metalloproteases can be down-regulated in the presence of the apoptotic-induced alterations, and that the decreased matrix-degrading capacity of this leukemic cell line during apoptosis may reduce its invasive potential.

Apoptosis↗

PubMed2000

Morphological features of apoptosis in hematopoietic cells belonging to the T-lymphoid and myeloid lineages.

Taking into account that apoptosis plays a pivotal role in shaping normal hematopoiesis, morphological features of apoptosis were investigated in both primary cells and continuous cell lines committed towards the T-lymphoid and the myeloid lineages. Apoptosis was induced using: dexamethasone (10(-7) M) for primary rat thymocytes; infection with the T-lymphotropic human herpesvirus 7 (HHV-7) for peripheral blood CD4+ T-cells; staurosporine (1 microM) for MOLT4 CD4+ lymphoblastoid T-cells, HL60 human promyelocytic and U937 human monoblastoid cells; and using senescence of the culture for primary human megakaryocytes. Cell morphology was examined by both transmission electron microscopy and in situ nick translation (NT) revealed by laser scanning confocal microscopy. In spite of the use of different apoptotic agonists, the morphological aspects of apoptosis were similar within the T-lymphoid and the myeloid lineage. While chromatin condensation characterized the early apoptotic events in both lineages, late apoptoses were mainly characterized by further nuclear condensation in lymphoid cells and by production of micronuclei in myeloid cells. Moreover, NT analysis clearly showed that the micronuclei derived from HL60 undergoing apoptosis were composed of both degraded and intact DNA. Thus, T-lymphocytes and myeloid cells showed a lineage-related behavior characterizing the late morphological aspects of apoptosis.

Animals↗

PubMed2000

Behavior of nucleolar proteins during the course of apoptosis in camptothecin-treated HL60 cells.

By means of immunofluorescence and immunoelectron microscopy we have studied the fate of different nucleolar components during the apoptotic process in camptothecin-treated HL60 cells. We have found that RNA polymerase I disappeared while UBF was associated with previously described fibrogranular threaded bodies. In contrast, fibrillarin, C23/nucleolin, and B23/nucleophosmin remained detectable in granular material present amid micronuclei of late apoptotic cells. Double immunolabeling experiments showed colocalization of both C23 and B23 with fibrillarin. Immunoblotting analysis showed that UBF was proteolytically degraded, whereas fibrillarin, C23/nucleolin, and B23/nucleophosmin were not. These results may help explain the presence of anti-nucleolar antibodies seen in various pathological disorders.

Apoptosis↗

PubMed2000

Giant cell tumor of tendon sheath: a light and electron microscopic study.

A neoformation has been surgically withdrawn from third finger extensor tendon of the right hand of a 52 year male subject. Light (LM) and electron microscope (EM) observations from a number of tissue fragments allowed the identification of tumor nature, which appeared a giant cell tendon sheath. Moreover, some structural patterns have been described and compared to the previously reported cases. In areas of major cell density, parenchyma does not show lobular or gland-like organization; on the other hand, wide zones characterized by an amorphous matrix, progressively replacing collagen and containing elongated cells, are present. Giant multinucleated cells, mostly localized close to collagen bundles, can also be revealed. Unexpectedly, no foam cells appear and no phagocytized cell debris can be identified in giant multinucleated cells. Engulfed crystals are, differently, evidentiated by electron microscopy, both in mono- and multinucleated cell cytoplasm. Their electron density and their localization within cytoplasmic vacuoles suggest the presence of calcium. A correlation between giant cells and osteoclasts is then proposed. Multiple variously oriented centrioles support the possible mitotic genesis of multinucleated giant cells, which never show, on the other hand, fusion features. Siderosomes and residual bodies are also present. An unusual, diffuse thickening of nuclear lamina, only interrupted at nuclear pore level, is described and discussed.

Giant Cell Tumors↗

PubMed1999

Apoptosis and necrosis following exposure of U937 cells to increasing concentrations of hydrogen peroxide: the effect of the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide.

A 3-hr exposure of U937 cells to hydrogen peroxide (H2O2) followed by a 6-hr posttreatment incubation in fresh culture medium promotes apoptosis or necrosis, depending on the oxidant concentration. Addition of 3-aminobenzamide (3AB) during the recovery phase prevented necrosis and caused apoptosis. 3AB did not, however, affect the apoptotic response of cells treated with apogenic concentrations of H2O2. Cells exposed for 3 hr to 1.5 mM H2O2, while showing some signs of suffering, maintained a normal nuclear organization and good organelle morphology. At the biochemical level, the oxidant promoted the formation of Mb-sized DNA fragments and rapidly depleted both the adenine nucleotide and non-protein sulphydryl pools, which did not recover during posttreatment incubation in the absence or presence of 3AB. These results allow a novel interpretation of the concentration-dependent switch from apoptosis to necrosis. We propose that H2O2 activates the apoptotic response at the early times of peroxide exposure and that this process can be completed, or inhibited, during the posttreatment incubation phase. Inhibition of apoptosis leads to necrosis and can be prevented by 3AB via a mechanism independent of inhibition of poly(ADP-ribose)polymerase. As a corollary, the necrotic response promoted by high concentrations of H2O2 in U937 cells appears to be the result of specific inhibition of the late steps of apoptosis.

Adenine Nucleotides↗

PubMed1999

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. The presence and degree of apoptosis were investigated in myeloid (HL-60 and K562), T (Jurkat and MOLT-4), and B (CESS and Raji) lymphoid cell lines by using a variety of techniques-transmission electron and light microscopy, flow cytometry and DNA gel electrophoresis. The major achievement of this study is that hematopoietic cells respond to different chemical (staurosporin, tiazofurin, camptothecin) and physical (hyperthermia or hypothermia) stimuli by apoptosis in a lineage-related way. Moreover, with respect to the methods used to detect apoptosis, a strong correlation was observed between the presence of the hypodiploid peak determined by flow cytometry and the DNA laddering evaluated by gel electrophoresis, but both techniques failed to demonstrate the presence of apoptosis in some cases. We conclude that cells of different hematopoietic lineages mostly show a lineage-related behaviour in their apoptotic response to different stimuli, suggesting that the lineage commitment and the stage of differentiation can confer different sensitivities to specific apoptotic stimuli. Moreover, morphological techniques still represent the most reliable approach to detect apoptosis in hemopoietic cells.

Apoptosis↗

PubMed1999

Selective modulation of the cyclin B/CDK1 and cyclin D/CDK4 complexes during in vitro human megakaryocyte development.

Mammalian megakaryocyte development is characterized by a progressive accumulation of cells exhibiting a polylobated nucleus with a polyploid DNA content. In this study human megakaryocytes were obtained from CD34+ haemopoietic progenitors by in vitro liquid culture in the presence of 100 ng/ml of recombinant thrombopoietin (TPO). Ultrastructural examination of polyploid megakaryocytes showed the presence of a large number of centrioles, the breakdown of the nuclear envelope, and the progressive chromatin condensation, all aspects characteristic of mitosis. At both indirect immunofluorescence and Western blot analyses, cyclin B and its related cyclin-dependent kinase (CDK)1, which forms the mitosis promoting factor (MPF), showed an increased expression in maturating megakaryoblasts and megakaryocytes (day 8 of culture) with respect to freshly isolated CD34+ progenitors. This expression tended to decline in fully developed megakaryocytes (day 15 of culture). The amount of cyclin D and of the related CDK4, governing the G1 phase of the cell cycle, increased during megakaryocyte development, maintaining high levels of expression also in mature megakaryocytes. These results indicate that megakaryocyte polyploidization depends on a true, although incomplete, mitotic process, and that cyclin D/CDK4 probably plays a crucial role throughout megakaryocytopoiesis.

CDC2 Protein Kinase↗

PubMed1999

Control of neuron outgrowth by NMDA receptors.

The role of N-methyl-D-aspartic acid receptors (NMDARs) of glutamate on neuritogenesis was studied in cultured neurons of chick embryo spinal cord using the NMDAR non-competitive antagonist dizocilpine maleate (MK-801). No cell population was fully prevented from neuritogenesis by MK-801. Different aspects of neuritogenesis were quantitatively evaluated. Neurite initiation, elongation and branching were depressed by MK-801. Inhibition was dose-dependent and reversible. A loss of responsiveness of neuritogenesis to MK-801 was found during the second day of treatment at a concentration of 10 microM, but not at higher concentrations. Our findings support the idea that Ca2+ influx through NMDAR associated channels is one of the possible triggers of a cascade resulting in neuritogenesis. The effects of NMDAR blocking on neuritogenesis occurred before synaptogenesis, suggesting a role of excitatory aminoacids in neuron morphological differentiation at early stages of development. Scanning electron microscopy confirmed a reduction in neurite tree complexity in MK-801 treated cells and showed a production of filopodium-like processes in some of these cells.

Animals↗

PubMed1998

A multiple technical approach to the study of apoptotic cell micronuclei.

Apoptotic micronuclei have been studied, in different cell types, from a morphologic and functional point of view. Conventional electron microscopy, in various staining conditions, selective cytochemistry for DNA, and freeze fracture for the analysis of chromatin fiber organization and size were performed. In situ TdT and Pol I immunofluorescent techniques were carried out to detect double- and single-strand DNA breaking points by confocal laser scanning microscopy. Apoptotic cell ultrathin cryosections were also performed and were analysed by field emission in lens scanning electron microscopy. Double/single strand massively cleaved DNA was detected in micronuclei, with a highly supercoiled, uniformly packed, very dense arrangement.

Animals↗

PubMed1998

Phospholipid rearrangement of apoptotic membrane does not depend on nuclear activity.

The behaviour of plasma membrane was studied in UV-treated cells to investigate its involvement in apoptosis. It was studied in HL60 cells, in which DNA oligonucleosomic cleavage occurs, and in Molt-4 cells, which are characterised by a different fragmentation pattern. During the early stages of apoptosis, a membrane lipid rearrangement occurs, which involves phosphatidylserine translocation from the inner to the outer leaflet. This molecular alteration was investigated by annexin V-FITC binding, analysed by flow cytometry and confocal microscopy. It was correlated with transmission electron microscopy, subdiploid peak appearance and DNA fragmentation. Our data indicate that the plasma membrane represents an early apoptotic target, even if its alterations are not detectable by ultrastructural analysis, which indicates its good preservation until late apoptotic stages. In addition, the study of apoptotic cells with absent or inactivated endonuclease demonstrates the independence of this membrane mechanism from nuclear activity.

Apoptosis↗

PubMed1998

Dimethylsulfoxide-induced cell death of murine erythroleukemia cells exposed to ionising radiation.

The present investigation was aimed at studying the effects of dimethylsulfoxide (DMSO) in combination with high dose (15 and 60 Gy) ionising radiation on the growth and differentiation of murine erythroleukemia cells (MEL). The incubation with DMSO was performed for 96 h starting immediately after exposure to radiation and resulted only in a slight inhibition of cell growth and in a high increase in cell death with the induction of both necrosis and apoptosis. The enhancement of radiation cytotoxicity was directly related to dose, time in culture and degree of differentiation as demonstrated by the severe and multiple aberrations observed in light and electron microscopy. Of interest was the observation in induced cells of a marked rearrangement of the plasma membrane architecture as well as that of the nuclear envelope, with a massive translocation and/or decrease in the nuclear pore complexes.

Animals↗