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Biomedical subjects

E Falcieri

Publications and source records attributed to E Falcieri.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1998

Ultrastructural changes in rat cornea after prolonged hypobaric hypoxia.

A study was performed to evaluate the effect of prolonged environmental hypobaric hypoxia on the ultrastructure of rat cornea. 60-day-old female Wistar rats were exposed to a simulated altitude of 5,500 m (350 mmHg) and pO2 of 76 mmHg for 30 days. Control rats were exposed to atmospheric sea level pressure (760 mmHg) and pO2 of 150 mmHg, for the same period. Ultrastructural analysis of the corneal epithelium did not reveal any relevant difference between control and treated rats. In contrast, the corneal stroma of rats subjected to hypoxia showed vascularization with advanced vessel differentiation and signs of active proliferation. The endothelium of hypoxic cornea showed swollen mitochondria and large empty cytoplasmic areas. The endothelial intercellular junctions could hardly be identified in the hypoxic condition. Nevertheless, the most evident change in hypoxic cornea was in Descemet's membrane, which was considerably thickened, to approximately twice that of the control specimen. These results suggest that environmental hypobaric hypoxia induces marked alterations in the corneal stroma and endothelium morphology, probably related to reduced oxygen tension in the aqueous humor, consequent to hypoxemia.

Animals↗
PubMed1998

The K562 chronic myeloid leukemia cell line undergoes apoptosis in response to interferon-alpha.

BACKGROUND AND OBJECTIVE: The K562 cell line, derived from a chronic myeloid leukemia (CML) patient and expressing B3A2 bcr-abl hybrid gene, is known to be particularly resistant to apoptotic death. IFN-alpha treatment of CML patients impairs malignant cell clone, apparently protecting from progression to terminal blast crisis. The mechanisms underlying this kind of cell deletion are analyzed here by multiple technical approaches. DESIGN AND METHODS: K562 cells, variably treated with IFN-alpha, were examined by agarose gel DNA electrophoresis, light and electron microscopy. The presence of bcr-abl rearrangement was revealed by RT-PCR. RESULTS: At 4 day treatment both DNA ladder and apoptotic nuclear changes were identified, consistently in the presence of bcr-abl expression. INTERPRETATION AND CONCLUSIONS: Even cells expressing bcr-abl, such as K562, can be triggered to apoptosis. Therefore, this genetic condition, commonly preventing PCD, does not prevent IFN-alpha-mediated apoptosis. PCD seems thus to be the mechanism underlying IFN-alpha-treated K562 cell deletion and it could be the basis of malignant clone reduction in IFN-alpha treated CML patients.

Apoptosis↗
PubMed1997

Human Herpesvirus 7 induces CD4(+) T-cell death by two distinct mechanisms: necrotic lysis in productively infected cells and apoptosis in uninfected or nonproductively infected cells.

We have investigated the cytopathic effects induced by the T-lymphotropic human herpesvirus 7 (HHV-7) on the CD4(+) T-lymphoblastoid SupT1 cell line and primary CD4(+) T lymphocytes. Acute in vitro HHV-7 infection induced (1) the formation of giant multinucleated syncytia, which eventually underwent necrotic lysis, and (2) single-cell apoptosis. Both cytopathic effects increased with the progression of infection and were blocked by phosphonoformic acid, a specific inhibitor of herpetic DNA polymerase. Using electron microscopy analysis of various samples, we found that all syncytia contained large amounts of virions and that most of them exhibited clear evidence of necrosis, whereas apoptosis was predominantly observed in single cells. Although empty viral capsids could be identified in the cytoplasm of approximately 25% of single cells exhibiting an apoptotic morphology, mature virions were hardly observed in these cells. In both coculture and cell-free HHV-7 infection experiments, a significant correlation was observed between the degree of single-cell apoptosis, evaluated by quantitative flow cytometry after propidium iodide staining, and the decrease in the total number of viable cells. Moreover, in cell-free infection experiments, apoptosis showed a positive correlation also with the viral load, monitored by quantitative HHV-7 DNA polymerase chain reaction. Thus, it appears that apoptosis occurred predominantly in uninfected bystander cells but not in productively HHV-7-infected cells.

Apoptosis↗
PubMed1997

In vitro senescence and apoptotic cell death of human megakaryocytes.

To investigate the fate of human megakaryocytes, CD34+ hematopoietic progenitor cells were purified from the peripheral blood or bone marrow of healthy donors and seeded in serum-free chemically defined suspension cultures. In the presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived cells showed an eightfold numerical expansion and a progressive maturation along the megakaryocytic lineage. Megakaryocyte maturation was characterized ultrastructurally by the presence of a demarcation membrane system and phenotypically by a high surface expression of alpha(IIb)beta3 integrin. The number of mature megakaryocytes peaked at days 12 to 15 of culture. On the other hand, the number of platelets released in the culture supernatant by CD34-derived megakaryocytes peaked at days 18 to 21, when a high percentage of megakaryocytes showed the characteristic features of apoptosis, as evaluated by electron microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end-labeling technique (TUNEL) and uptake of propidium iodide. In other experiments, primary alpha(IIb)beta3+ megakaryocytic cells were directly purified from the bone marrow aspirates of normal donors and seeded in serum-free suspension cultures. In the absence of cytokines, alpha(IIb)beta3+ megakaryocytes progressively underwent apoptotic cell death. The addition of TPO but not interleukin-3 or erythropoietin showed some protection of alpha(IIb)beta3+ cells from apoptosis at early culture times (days 2 to 4), but it did not show any significant effect at later time points. These findings suggest that the terminal phase of the megakaryocyte life span is characterized by the onset of apoptosis, which can be modulated only to a certain extent by TPO.

Antigens, CD34↗
PubMed1997

ATP depletion inhibits glucocorticoid-induced thymocyte apoptosis.

In quiescent thymocytes, mitochondrial de-energization was not correlated to apoptotic death. In fact, thymocytes treated with oligomycin, a highly specific inhibitor of ATP synthase, alone or with atractyloside to block ATP translocation from the cytoplasm, were alive, even if their mitochondria were depolarized, as revealed by flow cytometry after Rhodamine 123 staining. Furthermore, oligomycin was a powerful inhibitor of apoptosis induced in rat thymocytes by dexamethasone and, to a lesser extent, by the calcium ionophore A23187 and etoposide, but was without effect when apoptosis was induced by staurosporine, and increased cell death in mitogen-treated thymocytes. The inhibition of apoptosis was confirmed by morphological criteria, inhibition of inter-nucleosomal DNA fragmentation and inhibition of the loss of membrane integrity. The anti-apoptotic effect of oligomycin in cells treated with A23187 or etoposide was correlated to the inhibition of protein synthesis, while inhibition of apoptosis induced by dexamethasone, already evident at an oligomycin concentration of 10 ng/ml, was instead strictly correlated to the effect exerted on the cellular ATP level. Thymocyte apoptosis triggered by dexamethasone was blocked or delayed by inhibitors of respiratory-chain uncouplers, inhibitors of ATP synthase and antioxidants: a lasting protection from dexamethasone-induced apoptosis was always correlated to a drastic and rapid reduction in ATP level (31-35% of control), while a delay in the death process was characterized by a moderate decrease in ATP (73-82% of control). Oligomycin inhibited the specific binding of radioactive corticosteroid to thymocyte nuclei, confirming the inhibitory effect of ATP depletion on glucocorticoid binding and suggesting that ATP depletion is a common mediator of the anti-apoptotic action of different effectors in glucocorticoid-induced apoptosis. In conclusion, the reported data indicate that ATP may act as a cellular modulator of some forms of apoptosis, depending on the death trigger, and that in quiescent cells the de-energization of mitochondria is not necessarily linked to apoptosis.

Adenosine Triphosphate↗
PubMed1997

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.

Apoptosis↗
PubMed1997

Changes in the subnuclear distribution of two RNA metabolism-related proteins can be detected in nuclear scaffold or matrix prepared by different techniques.

The nuclear scaffold or matrix is a mainly proteinaceous structure thought to act as a nucleoskeleton determining the higher order organization of eukaryotic chromatin. These structures are prepared from isolated nuclei by a series of extraction steps involving the use of ionic detergents or high salt, and restriction enzymes or non-specific nucleases to remove chromatin and other loosely bound components. Since these treatments are harsh and unphysiological, the question remains open as to whether or not these structures, isolated in vitro, correspond to a nucleoskeleton existing in vivo. Recently, it has been demonstrated that the majority of nuclear matrix proteins are involved in RNA metabolism. In this study we have employed a morphological approach involving the use of confocal laser scanning microscopy and indirect immunofluorescence techniques to analyze whether two widely employed methods to prepare the nuclear scaffold or matrix can maintain the spatial distribution of two polypeptides involved in RNA metabolism, i.e., a 105-kDa component of spliceosomes and a ribonucleoprotein antigen. We demonstrate that the localization of these polypeptides changes, in some cases dramatically, in the final nucleoskeletal structures when compared with intact cells. Only when isolated nuclei were stabilized in vitro with the cross-linking agent sodium tetrathionate (NaTT) prior to extraction with 2 M NaCl and DNase I digestion, were the immunofluorescent patterns displayed by the nuclear matrix indistinguishable from those detected in intact cells. These results emphasize the usefulness of NaTT in studying putative nucleoskeletal structures, but also show that the methods currently employed to prepare the nuclear scaffold or matrix may create in vitro artifacts.

Blotting, Western↗
PubMed1997

Calcific chronic lateral epicondylitis: a histological and ultrastructural study.

Fragments of insertion tissue from right arm common extensor muscle have been collected from a 25-year patient with chronic lateral epicondylitis. Specimens, processed for light (LM) and electron (EM) microscopy, evidentiated a variety of degenerative alterations, such as focal hyalinosis, lipoidosis, collagen fiber redistribution, calcifications and vascular changes. Evidence of collagen normal function maintenance and turnover have been also observed in tenocytes.

Adult↗
PubMed1996

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.

Antibodies, Monoclonal↗
PubMed1996

Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation.

By flow cytometry, we have quantitatively evaluated HL-60, MOLT-4, and P815 apoptotic cells induced with camptothecin, staurosporine, and hyperthermia, respectively. Apoptosis was measured by two different flow cytometry techniques, using propidium iodide (PI) uptake in permeabilized and nonpermeabilized cells. Apoptosis also has been analyzed by electron microscopy and DNA cleavage by in situ nick translation and DNA gel electrophoresis. We demonstrate that supravital exposure to propidium iodide without prior permeabilization identifies apoptotic cells, and clearly distinguishes them from necrotic cells in all the cases examined. This capability is independent of the nucleosomal (180-200 bp) fragmentation of DNA (which does not take place in MOLT-4 cells), which is the basis for detection of apoptosis both as a "ladder" by DNA gel electrophoresis and as a hypodiploid peak by flow cytometry. Therefore, alterations in membrane permeability, on which PI uptake in living cells is based, allow distinction of apoptotic cells from necrotic and living cells independently of the heterogeneous biochemical patterns involved in programmed cell death, which may or may not lead to DNA oligonucleosomal fragmentation.

Animals↗
PubMed1996

Morphological patterns and DNA polymerase regulation in apoptotic HL60 cells.

An ultrastructural and functional study was performed during methotrexate (MTX) induced apoptosis on HL-60 leukemia cells. The fine preservation of plasma membrane architecture and organellar components was present until latest apoptotic stages, despite strong nuclear changes. This observation suggests the presence of a residual cell metabolic activity, which is here investigated. DNA agarose gel electrophoresis demonstrated its fragmentation, which was also confirmed in situ by nick translation confocal analysis. Nuclear purification was subsequently performed to investigate DNA polymerase activities. DNA polymerase alpha activity appeared strongly reduced from the early phases of methotrexate treatment, while the rate of DNA polymerase beta synthesis was found to increase progressively along with the time of drug treatment. Low levels of DNA polymerase gamma activity revealed both in control and in treated cells, suggesting the irrelevant involvement of this enzyme in the DNA metabolism of this model. DNA polymerase beta appears thus to be the enzyme preminently correlated to cell attempts to repair the methotrexate-induced DNA damage.

Apoptosis↗