PubMed Health⌕ Search

Biomedical subjects

F Luchetti

Publications and source records attributed to F Luchetti.

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↗
PubMed2001

Disassembly of nuclear bodies during arousal from hibernation: an in vitro study.

In previous studies we demonstrated that during hibernation cell nuclei contain structural constituents usually absent in euthermia. The rapid disappearance of such nuclear bodies upon arousal makes very difficult the in vivo investigation of the disassembly process, which could clarify their functions in nuclear metabolism in the hibernator. In the present study we subjected liver samples taken from hibernating edible dormice ( Glis glis) to different in vitro experimental conditions: at 4 degrees C, to preserve the hypothermic state of the hibernating organism; at 37 degrees C, to simulate the drastic increase in body temperature occurring during arousal; at 37 degrees C, in culture medium containing 10(-5) M delta opioid D-Ala2- D-Leu5 enkephalin, which mimics the activity of the hibernation induction trigger in hibernators. Electron microscopic analysis of hepatocyte nuclei at increasing incubation times revealed the subsequent steps of disassembly of coiled bodies, amorphous bodies and fibro-granular material, the unusual structural constituents accumulating during hibernation in these nuclei. We demonstrated that: (1) a temperature of 37 degrees C induces the disappearance of all nuclear bodies typical of hibernation in a few minutes; (2) both low temperature and hibernation-triggering opioid are able to slow down, although to different extents, the process of disassembly of nuclear bodies; (3) the fibro-granular material rapidly disappears during the early phases of incubation; while (4) coiled bodies and amorphous bodies progressively disassemble as fibrous material. Our results support previous hypotheses based on in vivo observations about a possible role for coiled bodies, amorphous bodies and fibro-granular material as storage/assembly sites of molecules needed for the rapid and massive resumption of transcriptional and post-transcriptional activities upon arousal and suggest a strict correlation between the dynamics and metabolic rate of nuclear bodies.

Animals↗
PubMed2001

Supravital exposure to propidium iodide identifies apoptosis on adherent cells.

BACKGROUND: Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.

Annexin A5↗
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

Prostate-specific antigen synthesis and secretion by human placenta: a physiological kallikrein source during pregnancy.

Prostate-specific antigen (PSA), a kallikrein-like serine protease until recently thought to be prostate specific, has been demonstrated in various nonprostatic tissues and body fluids. PSA has been also found in human endometrium and amniotic fluids, even if the significance of this novel expression is unclear. In this study, we have demonstrated by multiple techniques that human placental tissue, obtained at delivery from normal full-term pregnancies, synthesizes and secretes PSA. RT-PCR showed the presence of PSA messenger ribonucleic acid; biochemical, chromatographic, and immunological studies revealed the expression of both free and complexed PSA forms; immunoelectron microscopy indicated the syncytiotrophoblast as the site of PSA synthesis and secretion. Moreover, in vitro experiments demonstrated that PSA production and secretion are up-regulated by 17beta-estradiol, a pregnancy-related steroid hormone. These results suggest that human placenta is a source of the PSA present in amniotic fluid and maternal serum during pregnancy.

Adult↗
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↗
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

Immunoreactivity, ultrastructural localization, and transcript expression of prostate-specific antigen in human neuroblastoma cell lines.

Prostate-specific antigen (PSA) is considered a highly specific biochemical marker of the human prostate gland, and it currently is used for prostate cancer diagnosis and monitoring. Recently, PSA production and secretion were found in nondiseased and diseased cells, tissues, and fluids from women. In this study, we characterized the presence of PSA in two human neuroblastoma cell lines with biochemical, ultrastructural, and molecular approaches. Using reverse transcription-PCR, we identified PSA mRNA, and Western blotting revealed a substantial amount of complexed form of PSA protein, which is localized mainly in free ribosomes. Although the role of PSA in human neuroblastoma cell lines is still unknown, our study supports the hypothesis that this serine protease may be involved in controlling the growth of human brain tumor cells, adding more support to the notion that PSA is a widespread kallikrein-like protease with biological functions much more complex than recently thought.

Blotting, Western↗
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

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↗