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Biomedical subjects

C Pellicciari

Publications and source records attributed to C Pellicciari.

At least 37 records · Page 2Linked to original sources
PubMed1999

Growth and differentiation of the vascular smooth muscle and endothelial cells cultured on fluorine ion-implanted polystyrene.

The rat vascular (SMCs) and bovine endothelial cells (BECs) were cultured on conventional or fluorine ion-implanted polystyrene (5 x 10(12) and 5 x 10(14) fluorine ions/cm2). The cells grown on the implanted growth supports showed better adherence, higher volume and higher total protein content. The immunocytochemical analysis revealed that SMCs contained more of the cytoskeletal vimentin and the vascular SMC-specific alpha-actin as well as several cell adhesion-mediating molecules (vinculin, talin, alpha(v)-integrin and ICAM-1). In BECs, only the content of vimentin and talin increased, while expression of ICAM-1 was unchanged. The data suggest that cells on the ion implanted polymers could be more viable and that increased expression of some adhesion molecules mediating interactions with the host immune system is cell type-dependent.

Actins↗
PubMed1999

Different responsiveness of male and female rat aortic smooth muscle cells (SMCs) to repeated passaging in culture.

The smooth muscle cell (SMC) cultures were prepared from the aorta of male and female 8-week-old rats and used at passage 5-7 or 40-45. On day 1, low-passaged cells of both sex groups adhered to growth supports at similar numbers while after repeated passaging the adherence of female-derived cells was higher. These cells had also higher total protein content and contained more of the SMC specific alpha-actin, vimentin and alpha(v) integrins. Compared to the male type of cultures, the high passaged cells of female origin cycled at a slower rate and were undergoing massive polyploidization. Male-derived cells remained of the same morphology, ploidy and the differentiation status at all passages. Their passage response consisted mainly in faster cycling and growth to higher population densities. The data could be of importance for explanation of different incidence of hyperplastic vascular diseases in males and females.

Actins↗
PubMed1998

Bivariate flow cytometric analysis of DNA content versus immunopositivity for ribonucleotide reductase M1 subunit in the cell cycle.

Ribonucleotide reductase (RR) is a cytoplasmatic enzyme catalyzing the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides. Its activity strongly correlates to the rate of DNA synthesis. By using a specific monoclonal antibody against the large M1 subunit of RR, we assessed the expression of M1-RR versus DNA content by dual-parameter flow cytometry. The aim of this paper was to compare the variations in the immunopositivity for M1-RR during the cell cycle to the positivity for other cell cycle markers identifying either proliferating cells (Ki-67 and PCNA) or quiescent cells (statin). To do this, normal human embryonic fibroblasts in different growth conditions as well as several other mammalian cell lines (rat C6 glioma cells; mouse 3T3 fibroblasts and B16 melanoma cells; human epithelial EUE cells and mammary carcinoma MCF-7 cells) were used. The expression of M1-RR antigen was found to correlate positively with the expression of Ki-67 and PCNA, and negatively with the expression of statin. During early G1 phase, M1-RR becomes detectable by specific antibodies relatively later compared to PCNA and Ki-67; therefore, the lack of immunopositivity for M1-RR cannot be taken as an absolute indication of cell quiescence in G0.

3T3 Cells↗
PubMed1998

All-trans retinoic acid (ATRA)-induced apoptosis is preceded by G1 arrest in human MCF-7 breast cancer cells.

In this study the effects of all-trans retinoic acid (ATRA) on cell cycle and apoptosis of MCF-7 human breast cancer cells were investigated to elucidate the mechanisms underlying the antineoplastic potential of this retinoid in breast cancer. The antiproliferative effect of ATRA was evaluated by DNA content measurements and dual-parameter flow cytometry of bromodeoxyuridine (BrdU) incorporation and of the expression of cell cycle-related proteins (Ki-67 as proliferation marker and statin as quiescence marker) vs DNA content. Apoptosis was also studied by flow cytometry of either DNA content or Annexin V labelling. After 10(-6) M ATRA treatment, the fraction of S-phase cells decreased significantly, and cells accumulated in the G0/G1 range of DNA contents. Dual-parameter flow cytograms showed a decrease in the percentage of Ki-67-labelled cells (after 10 days, only 20% of the cells were still positive for Ki-67 compared with 95% in controls), while the fraction of statin-positive cells increased slightly. From 3 days of treatment onwards, apoptosis was found to occur. These results show that ATRA-induced inhibition of MCF-7 cell growth is related to two mechanisms, i.e. the block of cell proliferation, mostly in a pre-S phase, and the induction of apoptosis. These results should be taken into account when attempting to design treatment programmes that associate ATRA with antineoplastic compounds of different cell cycle specificity.

Annexin A5↗
PubMed1998

Nuclear RNA is extruded from apoptotic cells.

During spontaneous apoptosis of thymocytes there is extrusion of ribonucleoproteins (RNPs) from the cell. The aim of this investigation was to elucidate whether the RNP aggregates in apoptotic cells and bodies still contain RNA in an appreciable amount. We demonstrated by specific cytochemical techniques that the aggregates of nuclear RNPs extruded in the cytoplasm of spontaneously apoptotic thymocytes contain RNA in a sufficient amount to be detected cytochemically. These heterogeneous ectopic RNP-derived structures (HERDS) are formed by perichromatin fibrils, interchromatin granules, perichromatin granules, and nucleolar material. The RNA detected inside these clusters should therefore correspond to both mRNA and snRNA as well as to rRNA. We never observed DNA-containing aggregates in the cytoplasm of apoptotic thymocytes. The presence of RNA in the HERDS that may be released from apoptotic cells suggests that the decrease in the amount of total RNA during apoptosis may be mostly linked to cellular extrusion rather than to degradation of RNA by RNase activities. Another interesting aspect of these results lies in the hypothesis of apoptosis as a possible cause for the presence of autoantibodies in the serum of patients with systemic autoimmune diseases.

Animals↗
PubMed1998

LFA-1 mediated cell adhesion induces G0-G1 transition of human T lymphocytes in vitro.

Proliferation of mature T lymphocytes requires antigenic stimulation of the T cell receptor/CD3 complex (TCR/CD3) and an additional signal provided by different accessory molecules, including the leukocyte adhesion receptor LFA-1. We have used a cytochemical approach to analyse the effect of LFA-1 stimulation, either alone or in association with TCR/CD3 triggering. A dual parameter cytometric analysis of DNA content versus Ki-67 positivity allowed progression throughout the cell cycle to be monitored. Engagement of LFA-1 alone was able to initiate the intracellular events necessary for Ki-67 expression (marking G0-G1 transition) in a fraction of the T cell population but was not sufficient to induce the transit into S-phase. Cross-linking of both LFA-1 and CD3 was required for DNA synthesis to occur. These data confirm LFA-1 as an important costimulatory molecule of TCR-mediated T cell activation.

Antibodies, Monoclonal↗
PubMed1997

Nuclear ribonucleoprotein-containing structures undergo severe rearrangement during spontaneous thymocyte apoptosis. A morphological study by electron microscopy.

The rearrangement of nuclear ribonucleoprotein (RNP)-containing structures during spontaneous thymocyte apoptosis has been investigated by electron microscopy. Along with chromatin margination and condensation. RNPs segregate into the central portion of the nucleus, in the form of heterogeneous clusters of granules which contain interchromatin and perichromatin granules, perichromatin fibrils, nucleolar components, and probably coiled bodies. In parallel with progressive chromatin condensation and karyorrhexis, granule clusters are then extruded into the cytoplasm and are finally released at the cell surface as membrane-bound cytoplasmic debris, sometimes in association with apparently undamaged organelles such as centrioles. It is likely that this RNP segregation may correlate with a severe impairment of protein synthesis. A similar phenomenon was observed in elongating spermatids, when transcriptional arrest is induced during the process of reversible silencing of the male genome. It may be hypothesized that segregation into heterogeneous granule clusters could be a common mechanism to remove redundant RNP-containing structures from the cell.

Animals↗
PubMed1997

Dopaminergic phenotype induced by oestrogens in a human neuroblastoma cell line.

Oestrogens are the key factor in the sexual differentiation of the mammalian brain and play an important role in the activity of selected areas of the mature brain. To pursue the study of oestrogen action on neural cells at the molecular level, we developed a human neuroblastoma cell line (SK-ER3) expressing the oestrogen receptor (ER). Treatment of these cells with 17beta-oestradiol causes growth arrest and morphological and biochemical differentiation. The aim of the present study was to investigate whether oestrogen-differentiated SK-ER3 neuroblastoma cells acquire the ability to synthesize a specific neurotransmitter and whether the growth arrest previously reported can be ascribed to the blockage of the cells at a specific stage of the cell cycle. The results presented here indicate that oestrogens induce accumulation of SK-ER3 cells in the G0 phase of the cell cycle, underscoring the acquisition of a mature neural phenotype upon hormonal treatment. Most importantly, we show that in the differentiated cells the content of tyrosine hydroxylase and Na+-dependent dopamine uptake is significantly augmented, proving that the oestrogen-differentiated SK-ER3 cells can synthesize and store a specific neurotransmitter. In addition, we prove that the dopamine accumulated in differentiated SK-ER3 cells can be released. These studies therefore suggest that oestrogen treatment results in the acquisition of a fully functional dopaminergic phenotype of SK-ER3 cells. Ample evidence shows a link between dopaminergic neurons and oestrogen activity in hypothalamic and non-hypothalamic areas of the mammalian brain. Our study indicates that oestrogens might play a primary role in committing undifferentiated neuroblasts towards the dopaminergic phenotype.

Dopamine↗
PubMed1997

Occurrence of DNA sequence differences in C-heterochromatin of Eulemur coronatus and Eulemur macaco, as revealed by fluorescence resonance energy transfer.

In the genus Eulemur (Malagasy lemurs) karyotype diversification has occurred mainly through Robertsonian mechanisms of chromosome fusion (Rumpler et al., 1976). Eulemur coronatus is the sole species to have the largest genome size, due to a very large amount of C-heterochromatin, mostly located at the pericentromeric regions of the largest chromosomes (Warter and Rumpler, 1985). This increase in C-heterochromatin was thought to be due to DNA amplification (Ronchetti et al., 1993). The aim of this work was to investigate whether the large C-heterochromatin of Eulemur coronatus might have derived by amplification of the smaller C-heterochromatin of Eulemur macaco, a closely related species with smaller genome size. To obtain information on the overall base composition of the total genomes, on the relative interspersion of AT and GC base paris along the DNA molecule and on the structural differences in C-heterochromatin, we used a quantitative cyto-chemical approach, based on fluorescence resonance energy transfer (FRET) between DNA-specific fluorochromes (i.e. the AT-specific Hoechst 33258, and the non base-specific dye, propidium iodide). Micro-spectrofluorometry and image analysis were used to investigate both the overall FRET efficiency and its spatial distribution along the chromosome arms. FRET efficiency values of the DNA in C-heterochromatin were significantly different in the two Eulemur species, indicating a different qualitative composition of repetitive DNA. This suggests that the repetitive DNA of Eulemur coronatus cannot have originated by amplification in toto of the repetitive DNA sequences of Eulemur macaco.

Animals↗
PubMed1997

Detection of apoptotic cells by annexin V labeling at electron microscopy.

Annexin V is a Ca(++)-binding protein which is widely used as a marker for apoptotic cells, as it binds to the phosphatidylserine residues exposed at the surface of apoptotic cells. In this paper, we describe a method for the immunogold-labeling of biotin-conjugated Annexin V, to detect apoptotic thymocytes at electron microscopy. Etoposide-treated thymocytes were reacted in tissue culture medium with biotin-conjugated Annexin V, fixed with glutaraldehyde, and processed for resin embedding; thin sections were incubated with antibiotin antibodies coupled with colloidal gold. Cytometric estimates of the apoptotic index were also performed by evaluating either the DNA content after propidium iodide staining or the light-scattering values, as well as the positivity for fluorescein isothiocyanate-conjugated anti-biotin antibodies. At electron microscopy, gold-labeling of Annexin V was located on the plasma membrane only of apoptotic thymocytes and on cytoplasmic debris, likely resulting from the typical apoptotic blebbing. Unlabeled thymocytes always showed normal, non-apoptotic nuclear morphology. The application of Annexin V labeling at electron microscopy will allow a more refined description of the morphological events occurring during apoptosis.

Animals↗
PubMed1996

Spontaneous apoptosis of thymocytes is uncoupled with progression through the cell cycle.

Most developing lymphocytes spontaneously die in the thymus during positive and negative selection of the T cell repertoire. By evaluating the expression of the proliferation antigens Ki-67 and PCNA, we demonstrated here that more than 95% of thymocytes are potentially proliferating. The coincidence within the same cell population of death and proliferation is thus apparent in developing thymocytes. Using dual-parameter cytometric techniques to evaluate in single cells the amount of DNA versus light-scattering values, we found that spontaneous thymocyte apoptosis occurs with similar frequency in all the cycle phases, whereas apoptosis induced by the anti-topoisomerase-II, etoposide (which is the consequence of irreversible DNA damage), takes place with higher frequency in S and G2 phases (i.e., in those cycle phases in which DNA is subjected to torsional constraints). The capability of thymocytes to enter apoptosis was also monitored by digesting DNA in situ with DNase I (a nuclease that cleaves DNA mimicking the nuclear damage common to most apoptotic suicides). We also show that endonuclease-mediated DNA digestion occurs to a similar extent in cells with different DNA contents, i.e., in cycle phases in which the superstructural organization of chromatin is markedly different.

Animals↗
PubMed1996

Treatments with saline solutions and DNase I have different effects on DNA content and distribution in human and in mouse chromosomes.

The aim of this work was to re-examine, on a quantitative basis, the relationship between banding pattern after Giemsa staining and the amount (and distribution) of DNA along the length of the chromatid arms. To do this, we investigated by cytofluorometric methods the occurrence of possibly different extraction of chromosome DNA after some alternative G-banding procedure, i.e. treatment of chromosomes with saline solutions, or DNasi I digestion in situ. The G-banding procedure entailing trypsin pretreatment is known to be difficult to standardize; in the present investigation, it was also found that trypsin induced a massive, although quantitatively variable, extraction of DNA from fixed metaphase chromosomes. G-banding-like patterns may be obtained, by treating chromosomes preparations with saline solutions. Both PBS and Tris-HCl treatment for the times considered induced a G-banding-like pattern after Giemsa staining, regardless of the age of chromosome preparations; no banding was observed after staining DNA with PI, nor extraction of DNA was found to occur. DNase I digestion initially induced a G-banding in both human and mouse chromosomes after Giemsa staining, with concomitant extraction of DNA (but without apparent G-banding-like pattern after PI staining); after 30 min digestion, a C-banding-like pattern was observed after both Giemsa and PI staining. Exposure to PBS or Tris-HCl buffer at room temperature may therefore be recommended as a G-banding inducing treatment, since it allows the classification of single chromosomes after Giemsa staining, without determining significant displacement of genomic DNA, which can be submitted to further analysis in situ.

Animals↗
PubMed1996

Etoposide at different concentrations may open different apoptotic pathways in thymocytes.

Apoptosis of thymocytes has been investigated by morphological, biochemical and cytometric techniques, both in non-perturbed conditions and after exposure to the topoisomerase-II inhibitor etoposide in vitro. Apoptotic thymocytes were already present in untreated samples, and their frequency increased with increasing drug concentrations. Using flow cytometry, we demonstrated that the frequency of apoptotic thymocytes in S and G2 phase of the cell cycle was higher in etoposide-treated samples, consistent with the phase-preferential action proposed for this topoisomerase-II inhibitor. At electron microscopy, the sequence of nuclear events was apparently independent from the apoptogenic trigger, so that similar features of chromatin margination and nuclear fragmentation were observed in thymocytes undergoing apoptosis both spontaneously and after etoposide treatment. The morphology and function of plasma membrane were still preserved in apoptotic cells. At low (0.1 to 10 microM) etoposide concentrations apoptotic cells had cytoplasm patterns similar to naturally occurring apoptotic thymocytes, whereas at high (50 to 100 microM) concentrations extensive organelle damage took place. Damaged cytoplasm was sometimes polarized into discrete portions, which were eventually extruded within apoptotic bodies. This evidence suggests that, in the same cell type, different apoptotic pathways are opened by this agent, depending on the concentration used.

Animals↗
PubMed1995

Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content.

Statin, a 57-kDa nuclear protein, has been recognized as a unique marker of quiescent (G0) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of G0 cells by dual-parameter flow cytometry of statin expression and DNA content. The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen [PCNA] and Ki-67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S-44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual-parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF-7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNA and Ki-67, and with the BrdUrd labeling index. In dual-parameter flow scattergrams, G0 (statin positive) cells can be discriminated from the potentially cycling (statin negative) G1 cells, i.e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool both for monitoring changes in the resting cell fraction and for investigating the process of G0-G1 transition in unperturbed and drug-treated cell populations.

3T3 Cells↗
PubMed1995

Daudi lymphoma killing triggers the programmed death of cytotoxic V gamma 9/V delta 2 T lymphocytes.

A common feature of human V gamma 9/V delta 2 gamma delta T lymphocytes is their ability to kill Daudi lymphoma cells. We show here that during this killing a substantial fraction of cytotoxic gamma delta T cells dies as well. Their death has morphologic, cytometric, and biochemical features of apoptosis and depends on TCR triggering. We suggest that interaction with the target physiologically induces the programmed death of the cytotoxic gamma delta effector. The death of activated gamma delta T cells upon challenge with the relevant Ag may represent a negative feedback mechanism and contribute to explain the limited time span of gamma delta T cell expansions during infectious diseases, even when pathogens are not eliminated and persist in the host.

Apoptosis↗
PubMed1995

Cell cycle effects of hypertonic stress on various human cells in culture.

Long-term exposure to hypertonic (HT) culture media has been found to perturb the cell cycle and change gene expression in various animal cell types. A lower growth rate, with exit of cells from the cycling compartment has been observed previously in human transformed EUE cells. The aim of this study was to investigate if the kinetic changes after long-term HT stress, were typical of transformed cells or could be also found in primary cultures of normal cells. Human transformed cells from normal and neoplastic tissues, and normal human cells of epithelial and connective origin have been studied. After the incorporation of bromodeoxyuridine (BrdUrd), the frequency of S-phase cells was estimated by dual-parameter flow cytometry of DNA content versus BrdUrd immunolabelling; the total growth fraction was also estimated, after immunolabelling with an anti-PCNA antibody. We also investigated, by polyacrylamide gel electrophoresis, changes in the amount of a 35 kDa protein band, which increased in EUE cells grown in an HT medium, and which may be directly involved in cell resistance to hypertonicity. Lower BrdUrd labelling indices and higher frequencies of cells in the G0/1 range of DNA content were common features of all the cells in HT media, irrespective of their tissue of origin; other cycle phases may also be involved, depending on the cell type considered. The mechanisms by which cells cope with the HT environment could however differ, since only some cell types showed an increase of the 35 kDa stress protein found originally in HT EUE cells.

Cell Cycle↗