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M Piacentini

Publications and source records attributed to M Piacentini.

At least 91 records · Page 5Linked to original sources
PubMed1993

The role of apoptosis in growing and stationary rat ascites hepatoma, Yoshida AH-130.

The occurrence of apoptosis and its contribution to growth-phase transitions in the rat ascites hepatoma Yoshida AH-130 have been evaluated. Apoptosis was observed by light microscopy in both exponentially-growing and stationary tumours as characteristic chromatin condensation and compacting of the cytoplasm, but the frequency of apoptotic bodies (apoptotic index) was four-fold higher in stationary than in logarithmic-growing tumours. Apoptosing cells exhibited strong immunocytochemical reactivity for 'tissue' transglutaminase, and transglutaminase activity was higher in stationary tumour cells. A ladder pattern of DNA fragmentation was revealed by agarose gel electrophoresis which was more pronounced in stationary tumours. The apoptotic bodies were either free or contained in vacuoles within otherwise normal tumour cells, suggesting active engulfment of dead cells by viable homologous cells. Such 'homophagic' disposal of the apoptotic bodies probably accounted for a component of the enhanced cell protein degradation previously observed in stationary AH-130 tumours (Tessitore L, Bonelli G, Cecchini G, Amenta JS, Baccino FM, Arch Biochem Biophys 1987; 255: 372-384). The data indicate that, in concurrence with changes in the cell proliferation rate, modulations of apoptosis play a substantial role in determining the net growth rate of such an anaplastic tumour as the Yoshida AH-130 hepatoma.

Animals↗
PubMed1993

Correlation between induction of lymphocyte apoptosis and prostaglandin E2 production by macrophages infected with HIV.

Several data indicate that HIV infection of antigen-presenting cells (APCs) and apoptosis of lymphocytes play important roles in pathogenesis of AIDS. We have recently demonstrated that prostaglandin E2 (PGE2) can cause thymocyte apoptosis in vivo. In the present study we have investigated the possibility that the intercellular contacts between HIV-infected APCs and lymphocytes could induce apoptosis in the latter population and that PGE2-production by HIV-infected APCs could be involved in the hypothesized phenomenon. Monocytes/macrophages separated from peripheral blood mononuclear cells (PBM phi) of healthy donors were infected in vitro and maintained in culture. PGE2 was promptly produced by HIV-infected PBM phi and values of PGE2 concentrations in supernatants over the background noninfected controls persisted for more than 2 weeks. HIV-infected PBM phi or cell-free supernatants from their cultures were then added to autologous uninfected lymphocytes and apoptosis was assessed by morphological criteria and by looking to the expression of tissue transglutaminase, one of the effector elements of the program of cell death. In both culture conditions the percentage of apoptotic lymphocytes was significantly increased in respect to values obtained in control cultures. When a cyclooxygenase inhibitor was added to the HIV-infected PBM phi cultures, the percentage of apoptotic lymphocytes was reduced at levels similar to those observed after cocultivation with uninfected PBM phi or exposure to supernatants from uninfected PBM phi. In addition, a substantial increase in apoptosis in lymphocytes from healthy donors was found following PGE2 treatment in vitro.

Antigen-Presenting Cells↗
PubMed1993

Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis).

Proliferating primordial germ cells (PGCs) isolated from mouse embryos soon after their arrival in the genital ridges would only survive in vitro at temperature of less than 30 degrees C (De Felici, M. and McLaren, A. (1983). Exp. Cell. Res. 144, 417-427; Wabik-Sliz, B. and McLaren, A. (1984). Exp. Cell. Res. 154, 530-536) or when co-cultured on cell feeder layers (Donovan, P. J., Stott, D., Godin, I., Heasman, J. and Wylie, C. C. (1986). Cell 44, 831-838; De Felici, M. and Dolci, S. (1991). Dev. Biol. 147, 281-284). In the present paper we report that mouse PGC death in vitro occurs with all the hallmarks of programmed cell death or apoptosis. We found that after 4-5 hours in culture many PGCs isolated from 12.5 dpc fetal gonads assumed a nuclear morphology and produced membrane bound fragments (apoptotic bodies) typical of apoptotic cells. In addition, PGCs in culture accumulated high level of tissue transglutaminase (tTGase; an enzyme that is induced and activated during apoptosis) and showed extensive degradation of DNA to oligonucleosomal fragments, which is characteristic of apoptosis. The physiological relevance of this mechanism of PGC death is supported by the finding that some PGCs undergoing apoptosis, as revealed by the high level of tTGase expression, were detected in the embryo. Most importantly, we show that the addition of stem cell factor (SCF) or leukemia inhibitory factor (LIF) to the culture medium, two cytokines known to favour PGC survival and/or proliferation in vitro, markedly reduced the occurrence of apoptosis in PGCs during the first hours in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Characterization of the transglutaminase-mediated large molecular weight polymer from rat liver; its relationship to apoptosis.

A large molecular weight sodium dodecyl sulfate (SDS)-insoluble polymer was isolated from rat liver and shown to be a product of transglutaminase activity by virtue of its high epsilon(gamma glutamyl)lysine content. Antiserum raised against this polymer crossreacted with another product of transglutaminase activity, the apoptotic envelope isolated from cultured hamster fibrosarcoma cells. The amino acid composition of isolated apoptotic envelopes and the SDS-insoluble polymer were found to be comparable, although not identical. Using the polymer antiserum, the apoptotic index (mg polymer protein per mg DNA) of normal and tumor tissue was determined and found to correlate with the associated transglutaminase activity. Localization of proteins involved in polymer formation in neonatal rat liver cells was found immunohistochemically to be in those cells undergoing apoptosis; these cells also stained selectively for transglutaminase. Several proteins from liver homogenate were found to crossreact with the antipolymer serum, notably proteins of 120, 80, 43 and 38 kDa.

Animals↗
PubMed1993

Galactose-specific receptor modulation related to the onset of apoptosis in rat liver.

The expression of the asialoglycoprotein receptor of hepatocytes and the galactose-specific receptors of non-parenchymal liver cells during the onset of apoptosis in liver of rats treated with lead nitrate was studied. During the involution of lead nitrate-induced hyperplasia in rat liver (occurring at 5 days after the injection) a significant increase of asialoglycoprotein receptor (ASGP-R) expression on hepatocytes coincided with the massive death by apoptosis of the same cells. The increase in the receptor expression was sustained by a large increase in the level of its specific mRNA. As a consequence of lead nitrate injection, we also detected a drastic change of the galactose-specific receptor expression and distribution on the surface of rat liver sinusoidal cells. However, the modulation of the receptor expression on the Kupffer cells did not parallel that observed for the ASGP-R: the peak of surface expression measured on hepatocytes always followed the one observed on Kupffer cells. Our data show a first evidence of a receptor modulation during the process of apoptosis. In fact, the entire carbohydrate recognition system of the liver is modulated during the onset of apoptosis induced by lead nitrate injection, but the pattern of modulation depends on the cellular types. We suggest that a physiological role for the hepatic carbohydrate recognition systems is related to the apoptosis of liver.

Animals↗
PubMed1992

Phenotype-specific "tissue" transglutaminase regulation in human neuroblastoma cells in response to retinoic acid: correlation with cell death by apoptosis.

Neuroblastomas in culture are characterized by the presence of 2 morphologically and biochemically distinct phenotypes (i.e., neural "N-type" and flat substrate-adherent "S-type") which undergo transdifferentiation. Human neuroblastoma SK-N-BE(2) cells differentiate toward a neural phenotype upon retinoic acid (RA) treatment. However, we recently showed that, during the RA treatment, a subset of SK-N-BE(2) cells undergo apoptosis; these cells specifically express a high "tissue" transglutaminase (tTG) level. This study was undertaken to investigate the cellular and molecular basis of the action of retinoic acid on apoptosis in human neuroblastoma cells. As a biochemical marker of the phenomenon we studied the tTG gene expression in the parental line SK-N-BE(2) and in 2 clones which stably express neuroblastic [BE(2)-M17] and substrate-adherent [BE(2)-C] features, respectively. Data showed a differential phenotype-specific regulation of tTG gene expression. In fact, RA treatment enhanced tTG expression and apoptotic index in the flat substrate-adherent variant, whereas, in cells expressing the neural phenotype, very low tTG expression and apoptosis were found. Northern-blotting analysis revealed that the substrate-adherent cells had a basal 3-fold higher level of tTG mRNA. An increase in tTG mRNA major transcript levels (3.7 kb) occurred within a few hours of exposure to RA in both the phenotypic variants. By contrast, tTG protein level was very low in the cell expressing the neuronal phenotype, even after prolonged exposure to RA. Immunohistochemical analysis indicated that tTG protein, in addition to mature apoptotic cells, was specifically localized in the flat substrate-adherent variant both in the wild-type and in the BE(2)-C clone. These findings suggest that the ability to undergo apoptosis in the neuroblastoma cells is associated with the expression of a non-neuronal neuroectodermal (substrate-adherent cells) immature phenotype.

Cell Death↗
PubMed1992

In vivo and in vitro induction of 'tissue' transglutaminase in rat hepatocytes by retinoic acid.

Tissue transglutaminase (tTG) expression was found to be induced in rat liver following in vivo retinoic acid (RA) treatment (Piacentini et al. (1988) Biochem. J. 253, 33-38). Here we show that the increased enzyme expression in rat liver is at least partially the result of the action of RA in parenchymal cells. In fact, (a) when hepatocytes are isolated from RA-treated animals their transglutaminase protein content is much higher than in similarly isolated control cells; (b) higher tTG protein level is also found by immunoelectronmicroscopy in the hepatocytes of the RA-treated rats as compared with the very low amount detected in the controls; (c) RA induces tTG in hepatocytes under culture conditions as well. One of the functions of tTG is to form a protein polymer in dying apoptotic cells by epsilon(gamma-glutamyl)lysine and, specifically gamma-glutamylpolyamine cross-links (Fesus et al. (1989) FEBS Lett. 245, 150-154). Noteworthy, after in vivo and in vitro RA-treatment we could not determine any increase (there was even a slight decrease) in the number of the cross-linked apoptotic envelopes. In keeping with this is the significant reduction of protein bound gamma-glutamylpolyamine detected in hepatocytes exposed to RA in culture. These findings suggest that the RA-induced tTG in parenchimal cells is an inactive form.

Animals↗
PubMed1992

The clearance of apoptotic cells in the liver is mediated by the asialoglycoprotein receptor.

Apoptosing cells are actively phagocytosed in parenchymal tissues, thus preventing the inflammatory reaction which could derive from their slow uncontrolled degradation. The molecular mechanisms by which an apoptotic cell is recognized and taken up are largely unknown. We propose that the recognition of apoptotic hepatocytes is mediated by the sugar recognition systems of the liver, particularly the asialoglycoprotein receptor (ASGP-R). The results presented here demonstrated the participation of ASGP-R in the removal of apoptotic parenchymal cells, and indicate a new perspective for the understanding of its physiological role.

Acetylgalactosamine↗
PubMed1992

Cycloheximide can rescue heat-shocked L cells from death by blocking stress-induced apoptosis.

Cultured mouse L cells undergo apoptosis upon 1 h heat shock at 43 and 45 degrees C. Morphologically characteristic apoptotic cells begin to appear soon after the shock. Immunohistochemistry with anti-transglutaminase antibody shows that in most treated cells the enzyme is induced. Its activation results in the formation of highly cross-linked detergent-resistant apoptotic bodies during recovery. Cycloheximide added during hyperthermic stress inhibits the appearance of apoptotic bodies, showing that heat-shock-induced apoptosis is dependent on protein neosynthesis. The analysis of colony-forming ability of heat-shocked L cells shows a survival of 5% at 43 degrees C and less than 0.02% at 45 degrees C. When protein synthesis is inhibited during heat shock the fraction of surviving cells increases to 23% at 43 degrees C and 0.9% at 45 degrees C. This suggest that part of the cells that die upon heat shock are not heavily damaged and would have survived in the presence of a block in protein synthesis.

Animals↗
PubMed1992

Expression of tissue transglutaminase in Balb-C 3T3 fibroblasts: effects on cellular morphology and adhesion.

Tissue transglutaminase is a cytosolic enzyme whose primary function is to catalyze the covalent cross-linking of proteins. To investigate the functions of this enzyme in physiological systems, we have established lines of Balb-C 3T3 fibroblasts stably transfected with a constitutive tissue transglutaminase expression plasmid. Several cell lines expressing high levels of catalytically active tissue transglutaminase have been isolated and characterized. Transglutaminase-transfected cells showed morphologic features quite distinct from their nontransfected counterparts. Many of the cells showed an extended and very flattened morphology that reflected increased adhesion of the cells to the substratum. Other cells, particularly those showing the highest levels of intracellular transglutaminase expression, showed extensive membrane blebbing and cellular fragmentation. The results of these experiments suggest that the induction and activation of tissue transglutaminase may contribute both to changes in cellular morphology and adhesiveness.

3T3 Cells↗
PubMed1992

Induction of apoptosis in thymocytes by prostaglandin E2 in vivo.

In vivo administration in mice of a synthetic analog of prostaglandin E2 (PGE2) caused a selective and dramatic decrease of CD4+CD8+ double-positive, CD3/T-cell-receptor-alpha beta(lo) cells in the thymus. This loss was corticosteroid-independent and not affected by Cyclosporin A. The disappearance of CD4+CD8+ thymocytes was strictly correlated with the induction of apoptosis inside the thymus as shown by morphological studies and by the induction of intracellular transglutaminase expression. Considering that PGE2 has been found to be produced by different cell populations inside the thymus, these results indicate that PGE2 may act as endogenous signals for apoptosis during T-cell differentiation.

16,16-Dimethylprostaglandin E2↗
PubMed1991

Degradation of cells dying by apoptosis leads to accumulation of epsilon(gamma-glutamyl)lysine isodipeptide in culture fluid and blood.

epsilon(gamma-Glutamyl)lysine isodipeptide, the end-product of proteolytic digestion of proteins cross-linked by transglutaminase, was detected in culture fluid of neonatal rat hepatocytes and plasma of adult rats. The concentration of the isodipeptide was significantly increased in both when high rate of apoptosis with phagocytosis of dying hepatocytes was produced either by epidermal growth factor in the culture or by lead nitrate-induced hyperplasia with subsequent involution in rats. Specific induction of tissue transglutaminase and the consequent formation of highly cross-linked protein envelopes in apoptotic cells have been previously demonstrated by us in both systems.

Animals↗
PubMed1991

"Tissue" transglutaminase is specifically expressed in neonatal rat liver cells undergoing apoptosis upon epidermal growth factor-stimulation.

We recently reported that activation of "tissue" transglutaminase (EC 2.3.2.13; tTG) in liver cells undergoing apoptosis determines extensive cross-linking of cellular proteins resulting in the formation of SDS-insoluble shells in the so-called "apoptotic bodies". In attempt to obtain further insight into the role played by tTG in apoptosis of liver cells, we investigated its expression in primary cultures of neonatal rat liver cells stimulated with epidermal growth factor (EGF). EGF-treatment of neonatal rat liver cells induces first hyperplasia of hepatocytes, followed by involution characterized by a high incidence of apoptosis. The proliferative phase of hepatocytes is paralleled by a 10-fold increase in tTG mRNA level, which is followed, during the phase of involution, by sequential increases in enzyme activity and levels of SDS-insoluble apoptotic bodies. tTG immunostaining at both the light- and electron-microscopic levels shows that the most intensive reaction is present in globular structures showing the typical morphological appearance of mature apoptotic bodies. In early apoptotic stages, tTG protein is localized in the perinuclear region of the cell. Intense immunostaining is also found in the apoptotic bodies present inside phagosomes within the cytoplasm of neighboring cells. This evidence confirms and extends our previous findings, indicating that tTG induction and activation specifically takes place in cells undergoing apoptosis, suggesting a key role for the enzyme in the apoptotic program.

Animals↗
PubMed1991

The expression of "tissue" transglutaminase in two human cancer cell lines is related with the programmed cell death (apoptosis).

The expression of "tissue" transglutaminase (tTG) in two human tumor cell lines (the cervix adenocarcinoma line HeLa-TV and the neuroblastoma cells SK-N-BE-2) was found to be in correlation with the rate of physiological cell death (apoptosis) in culture. We investigated the effect of retinoic acid (RA) and alpha-difluoromethylornithine (DFMO) in order to elucidate the relationship between tTG expression and apoptosis. RA led to a 6-fold increase of tTG activity in HeLa-TV cells and to a 12-fold increase in SK-N-BE(2) cells, which was paralleled in both cell lines by a proportional increase in the number of apoptotic bodies recovered from the cultures. On the contrary, DFMO determined a dramatic reduction of tTG expression and of the apoptotic index. Immunohistochemical analysis using an anti-tTG antibody showed that the enzyme was accumulated in both cell lines within typical apoptotic bodies. Immunocytochemistry and cell cloning of SK-N-BE(2) line demonstrated that tTG was absent in cells showing neurite outgrowth, indicating that the enzyme expression is not associated with neural differentiation, even though both phenomena are elicited by retinoic acid. On the whole, these data indicate that also in tumors tTG activation takes place in cells undergoing apoptosis. The enzyme is activated in apoptotic cells to form cross-linked protein envelopes which are insoluble in detergents and chaotropic agents. The number of insoluble protein envelopes as well as the N,N-bis(gamma-glutamyl)polyamine cross-links is related with both tTG expression and apoptotic index, strongly suggesting the participation of the enzyme in the apoptotic program.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Survival↗
PubMed1991

Induction of two different modes of cell death, apoptosis and necrosis, in rat liver after a single dose of thioacetamide.

A sequential study of the appearance of liver cell death after thioacetamide (TH) administration was performed in male Wistar rats. Within 3 hours of a single dose of TH, occurrence of cell death by apoptosis was evident around the centrilobular area. Light as well as electron microscopic examination demonstrated the presence of eosinophilic globules, often containing nuclear remnants (apoptotic bodies); they frequently were found within the cytoplasm of intact hepatocytes. The number of apoptotic bodies (ABs) was further enhanced at 6 hours, resulting in a 70-fold increase over the control values. Although necrosis or inflammation could not be observed at this time, as monitored by microscopic analysis as well as by determination of serum glutamate pyruvate transaminase levels, centrilobular necrosis accompanied by massive inflammatory reaction was evident at 12 hours and even more pronounced at 24 to 36 hours. Evidence of liver regeneration was found to occur at 48 hours, and the liver regained its normal architecture between 72 and 96 hours. Studies performed to analyze the activity of 'tissue' transglutaminase (tTG), a presumptive marker of apoptosis, showed that, 1 hour after treatment, TH caused a drastic dose-dependent inhibition of the enzyme activity. This early inhibition was followed by a rapid recovery in tTG activity that paralleled the induction of apoptosis in the liver. Treatment with cycloheximide (CH) 2 hours after TH partially inhibited the incidence of ABs at 6 hours (approximately 30% inhibition). The present study indicates that two different modes of cell death, apoptosis and necrosis, may be induced in a sequential fashion by a single dose of TH.

Alanine Transaminase↗