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F A Manzoli

Publications and source records attributed to F A Manzoli.

At least 37 records · Page 2Linked to original sources

Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry.

Natural killer (NK) cells bind to K562 tumor target cells in vitro and kill them. The binding and cytotoxic activities of NK cells are tightly related to each other: degranulation of the cytotoxic effector is the basis for target cell damage and a consequence of effector-target recognition and binding. However, the two phases of NK activity, binding and killing, have always been measured separately by various methodologies and under different experimental conditions, because of the lack of a comprehensive methodology able to measure both of them at one time. Here we describe the simultaneous measurement of the binding and killing activities against K562 of resting and cytokine (IL-2 or IL-12)-stimulated NK cells by flow cytometry. NK, K562 and conjugates can be identified and measured by flow cytometry on the basis of NK mAb staining and target cells autofluorescence (Binding Plot). Within each population of the binding plot, killed targets can be identified and measured by their scatter characteristics (Cytotoxicity Plot). We show that i) the conjugate formation is enhanced in cytokine-stimulated cells, even at relatively short co-incubation times; ii) the conjugate release is also accelerated by cytokines; iii) the conjugate release is always quicker than the induction of the morphological changes in the target cell that generate its modified scattering properties.

Cell Line↗

Inositides in the nucleus: taking stock of PLC beta 1.

The nucleus was shown to be a site for inositol lipid cycle which can be affected by treatment of quiescent cells with growth factors such as IGF-I. In fact, the exposure of Swiss 3T3 cells to IGF-I results in a rapid and transient increase in nuclear PLC beta 1 activity. In addition, several other reports have shown the involvement of PLC beta 1 in nuclear signalling in different cell types. Indeed, PLC beta 1 differs from the PLC gamma and della isozymes in that it has a long COOH-terminal sequence which contains a cluster of lysine residues that are critical for association with the nucleus. Although the demonstration of PtInsP and PtdInsP2 hydrolysis by nuclear PLC beta 1 established the existence of nuclear PLC signalling, the significance of this autonomous pathway in the nucleus has yet to be thoroughly clarified. By inducing both the inhibition of PLC beta 1 expression by antisense RNA and its overexpression we show that this nuclear PLC is essential for the onset of DNA synthesis following IGF-I stimulation of quiescent Swiss 3T3 cells. Moreover, using a different cell system, i.e. Friend erythroleukemia cells induced to differentiate towards erythrocytes, it has been evidenced that there is a relationship between the expression and activity of nuclear PLC beta 1 and the association of PI-PT alpha with the nucleus in that, when PLC activity ceases, in differentiated and resting cells at the same time there is a dramatic decrease of the association of PI-PT alpha with the nucleus.

3T3 Cells↗

Cytochemistry of the functional domains of the nucleus in normal and in pathologic conditions.

By means of ultrastructural cytochemistry significant advances have been made in understanding the functional roles of many nuclear domains. This review gives schematic information about the main nuclear domains involved in replication, transcription, processing and transport of the transcripts in normal and in pathologic conditions. Particular attention is paid to a functional domain that appears to be involved in signal transduction. Data are reported on the intranuclear specific localization of key elements of the polyphosphoinositide signal transduction system in different cell types including human osteosarcoma cell lines. Compared with the compartmentalization of the cytoplasm, the nucleus has long been considered as relatively unstructured. On the other hand, fundamental nuclear functions, such as DNA replication and RNA transcription, can be molecularly characterized also in cell-free systems, suggesting that supramolecular organization is not so strictly required as for other cell functions occurring within intact cytoplasmic organelles. Nevertheless, a stringent organization is required for packing about 200 cm of DNA in the about 30 micron 3 of the nucleus. In the absence of membrane-delimited organelles, the nuclear organization is based on functional compartments, or domains, whose spatial localization involves the nuclear matrix, which shares many properties with the cytoskeleton. The nuclear domains are defined as structural compartments, not necessarily stable but dynamically variable, which perform specific metabolic functions through the partitioning of molecular complexes. Their identification has been made possible in the last few years by the development of specific nuclear probes for confocal and electron microscope immunocytochemistry. Therefore, the complex network of structures and enzymatic functions that make up the nucleus is in several cases yielding to molecular analysis, but a large part remains unknown (Strouboulis and Wolffe, 1996; Laemmli and Tjian, 1996). Rapid advances in understanding the functional role of the nuclear domains have been made recently: in particular, of the nuclear envelope, of the nucleolus, and of RNA splicing. In other cases, e.g. the precise localization of the nuclear domains involved in signal transduction, much remains to be clarified (Forbes and Johnson, 1997). It is conceivable that in the near future unexpected new nuclear domains will come to light and new nuclear functions may emerge, especially in field of post-transcriptional processing and transport of RNAs, and in the relationships between the nucleo-skeleton and enzymic fixed sites involved in replication, transcription and signal transduction. The aim of this review is to provide information about the morphological characteristics, the associated functions and the molecular composition of the main nuclear domains found to date. To simplify the exposition, the main data on each nuclear domain are reported in Tables, together with the principal references on the subject. Figures refer to original findings on some aspects of nuclear domain organization.

Cell Nucleus↗

Nuclear lipid-dependent signal transduction in human osteosarcoma cells.

The enzymes and substrates involved in phosphoinositide signal transduction which have been detected in the nucleus of several cell types have been demonstrated to be responsive to agonists. The complexity of this aspect of inositide function has been previously analyzed in some cell models characterized by a mitogenic or differentiating response to specific factors. An interesting experimental model is represented by human derived osteosarcoma Saos-2 cells, characterized by the expression of high affinity receptors for interleukin 1 alpha (IL-1 alpha), which is one of the most potent stimulators of bone resorption. In particular, we investigated the earliest intracellular events following the binding of IL-1 alpha to its receptor, involving the inositide signal transduction pathway. Saos-2 cells present a partitioning of the phosphoinositidase (PLC) isoforms; in fact, the nucleus contains both PLC beta 1 and gamma 1, while the cytoplasm contains almost exclusively the gamma 1 isoform. IL-1 alpha evokes a rapid and transient increase of the PLC beta 1 activity in the nucleus, which causes the hydrolysis of phosphatidylinositol mono- and bis-phosphate. In response to IL-1 alpha, not only the canonical inositol lipid pathway appears to be involved; also the 3'-phosphorylated lipids generated by phosphatidylinositol 3-kinase (PI 3-K), which may act as second messengers, appear to be affected. In fact, Saos-2 cells present a nuclear PI 3-K activity which can be enhanced by the IL-1 alpha treatment. Among the possible targets of the second messengers released by the nuclear PLC beta 1 activation, we found that some protein kinase C isoforms, namely the epsilon and zeta, which are present within the nucleus, are activated after IL-1 alpha exposure. These activated PKC isoforms, in turn, could modulate the activity of the transcription factor NFkB, which, 5 min after IL-1 alpha treatment, has already translocated to the nucleus and bound to DNA to promote gene activation. The actual role of the inositide pathway in the Saos-2 cell function has also been investigated by utilizing cell clones transfected with the mouse sequence of the PLC beta 1.

Animals↗

Selective enrichment in human megakaryocyte progenitors by the B203.13 surface differentiation antigen.

The B203.13 monoclonal antibody specifically recognizes a subset (8-18.5%) of human CD34+ haemopoietic progenitors, which are significantly (P<0.05) enriched in megakaryocyte progenitors (CFU-meg). Moreover B203.13 expression was progressively lost as maturation proceeded along the megakaryocytic lineage. In fact: (i) CFU-meg derived from CD34+/B203.13+ showed a greater number of cells/colony with respect to CD34+/B203.13-; (ii) after 10 d of liquid cultures with 100 ng/ml of thrombopoietin, only a minority of CD34-derived cells positive for CD41 coexpressed B203.13; (iii) in situ immunocytochemical staining revealed that B203.13 was expressed exclusively on immature megakaryoblasts; (iv) circulating platelets did not express B203.13.

Antigens, Differentiation↗

Stimulation of nuclear polyphosphoinositide synthesis by GTP-gamma-S: a potential regulatory role for nuclear GTP-binding proteins.

The nonhydrolyzable GTP analogue GTP-gamma-S was capable of stimulating in vitro phosphorylation of polyphosphoinositides in isolated nuclei prepared from mouse erythroleukemia cells. On the contrary, GDP-beta-S was ineffective. The stimulation was not detectable when nuclei were prepared from erythroleukemia cells induced to differentiate by exposure to dimethyl sulfoxide. Both nuclear phosphomonoesterase and phospholipase C activities were not influenced by GTP-gamma-S. Our results point to the likelihood that nuclear phosphoinositide kinases might be regulated by a GTP-binding protein.

Animals↗

Inositol lipid cycle and autonomous nuclear signalling.

The involvement of phospholipids and in particular polyphosphoinositides in cellular signalling has been documented in detail in the last 20 years. In addition to the plasma membrane localization also the nucleus is shown to be a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. Previous observation have established that the nucleus possesses a specific PLC for inositol lipids, i.e., the PLC beta 1 isoform, which undergoes rapid and transient activation after IGF-I stimulation of quiescent Swiss 3T3 cells and is down-regulated after treatment of Friend erythroleukemia cells with DMSO. Here we have reviewed: (i) the potential of nuclear PLC beta 1 to be a target for anti-cancer drug, (ii) the capability of this PLC isoform, when activated by IGF-I, to be a key signalling molecule in the onset of DNA synthesis, via DAG generation and PKC alpha translocation to the nucleus, (iii) the chromosome mapping of PLC beta 1 gene. The differentiation program of Friend cells can be activated by other agents besides DMSO including tiazofurin, an anti-tumor drug, also capable of affecting the nuclear inositol lipid cycle. Tiazofurin induces a lowering of the activity of PLC beta 1 due to down regulation of this isoform as revealed by both Western blotting and Northern blotting analyses. Using Swiss 3T3 cells stably transformed with an antisense PLC beta 1 construct, the knock-out of the PLC beta 1 gene induces both a loss of PLC beta 1 expression, as determined by Western blots, and a loss of the mitogenic responsiveness to IGF-I. These events show a direct relationship between nuclear PLC beta 1 evoked signals and IGF-I induced cell proliferation. Finally, the assignment of the PLC beta 1 gene to the band q35-36 of rat chromosome 3 paves the way for further genetic studies given the fact that the region where PLC beta 1 gene maps is a hot spot for genetic alterations in a number of experimentally induced rat tumors. Taken as a whole, these results assign a key role to the regulation of nuclear PLC activity and expression both in growth-factor activated mitogenesis and in in vitro erythroid differentiation.

Antineoplastic Agents↗

Nuclear inositol lipid cycle and differentiation.

Previous investigations from our laboratory and others have shown the existence of an autonomous intranuclear inositide cycle endowed with conventional lipid kinases and PLC which in PC12 pheochromocytoma cells, human osteosarcoma SaOS-2 cells, rat liver and Swiss 3T3 cells is the isoform beta 1, which in the latter cells is activated upon IGF-I stimulation. The behavior of the nuclear inositol lipid cycle has been investigated in nuclei of Friend erythroleukemia cells. These nuclei possess both lipid kinases and PLC. The cycle upon treatment with differentiating agents (i.e., DMSO and tiazofurin) is characterized by an accumulation of polyphosphoinositides and a decrease of DAG due to down-regulation of a specific PLC. Indeed, even if both beta 1 and gamma 1 isoforms are present in these nuclei, when Friend cells undergo terminal erythroid differentiation only the PLC beta 1 isoform is down-regulated as shown by immunochemical and immunocytochemical analysis, by direct determination of enzymatic activity and in the presence of neutralizing monoclonal antibodies as well as by Northern blot for PLC beta 1 message, whilst the amount of PLC gamma 1 and its activity are unaffected by erythroid differentiation. In conclusion, the presence of a specific nuclear PLC whose activity and expression are down-regulated during differentiation of erythroleukemia cells points out a role for nuclear phosphoinositide signalling in the processes of cell differentiation and hints at the nuclear PLC beta 1 as an important step of the cycle in relation to the erythroid differentiative commitment of murine erythroleukemia cells.

Animals↗

Immunocytochemical detection of the intranuclear variations of phosphatidylinositol 4,5-bisphosphate amount associated with changes of activity and amount of phospholipase C beta 1 in cells exposed to mitogenic or differentiating agonists.

The intracellular localizations of phosphatidylinositol 4,5-bisphosphate (PIP2) and of its hydrolyzing enzyme phospholipase C (PLC; in this case the beta 1 isoform) have been evaluated by electron microscope immunocytochemistry in cells exposed to mitogenic or differentiating agents. These cells have been previously demonstrated to present a signal transduction system based on the polyphosphoinositide hydrolysis localized at the nuclear level, which can be specifically modulated by agonists. The results demonstrate that in Swiss 3T3 mouse fibroblasts mitogenically stimulated by insulin-like growth factor I (IGF-I), a rapid and transient decrease of the PIP2 detectable by immunogold labeling occurs at the nuclear interior. This effect appears due to the activation of the PLC beta 1 isozyme already present in the nucleus, since no significant variations of the enzyme amount and distribution can be detected by immunolabeling. However, after 30 min of exposure to IGF-I, when the PLC beta 1 activity is returned to basal level, a slight but significant increase of the enzyme amount is detected both in the nucleus and in the cytoplasm. On the other hand, an increased accumulation of PIP2 in the nucleus, accompanied by a decrease of the intranuclear amount of PLC beta 1 isozyme, have been observed in mouse erythroleukemia Friend cells, induced to erythroid differentiation by dimethylsulfoxide (DMSO). These results indicate that quantitative immunocytochemistry represents an increment in the available methodologies to investigate the complex regulation of nuclear PI-signalling.

3T3 Cells↗

High resolution detection of human metaphase chromosomes.

The new generation of field emission in lens scanning electron microscopy with the sample located within the objective lens allows the observation of uncoated biological samples. Here we describe HeLa metaphase chromosomes isolated by two procedures and the localization within these chromosomes of a DNA centromeric probe. Chromosomes isolated in methanol acetic acid show 10 nm fibers organized in a three dimensional network while the structure of chromosomes obtained by the polyamine method's characterized by very dense short protusions of 30-50 nm in thickness. In situ hybridization with a DNA centromeric probe revealed by colloidal gold particles demonstrate that the hybridization occurs at the level of the 10 nm fiber with a high definition in the localization of specific DNA sequences.

Centromere↗

Lipid-dependent nuclear signalling: morphological and functional features.

Enzymes involved in lipid metabolism exist within the nucleus and are responsive to external stimuli. In particular, the kinases which phosphorylate phosphatidylinositol and phosphatidylinositol-4-monophosphate have been demonstrated in nuclei of both undifferentiated and differentiated Friend cells and of quiescent Swiss 3T3 cells as well as of those exposed to insulin-like growth factor I. Besides the lipid kinases, also the phosphoinositidases C (PIC) are active inside the nucleus. In Swiss 3T3 cells the nuclear PIC beta 1 is activated and its activation by IGF-I temporally precedes the translocation to the nucleus of protein kinase C. In Friend cell nuclei, on the other hand, when erythroid differentiation is induced, the PIC beta 1 activity is reduced. Another aspect of the nuclear signalling transduction system which appears quite interesting is its actual localization at subcellular level. By using electron microscope immunogold labelling, the nuclear PIC isoforms (the beta 1 isoform in Swiss 3T3 cells, the beta 1 and gamma 1 in Friend cells) are localized mainly in the interchromatin domains. This localization has been further confirmed on in situ matrix preparations of 3T3 cells in which PIC beta 1 is associated with the inner nuclear matrix but not with the nuclear pore-lamina complex. Colocalization experiments indicate that nuclear PIC beta 1 is present in sites in which both nuclear phospholipids and PKC can be detected, while the cytoplasmic PIC gamma 1 can be identified in close association with cytoskeletal filaments identified by anti-actin antibodies. The precise localization of the different PIC isoforms strongly indicates that the signal transduction system operating at the nuclear level may be part of a cross-talk between the cytoplasm and the nucleus controlling either cell proliferation or differentiation.

3T3 Cells↗

Phosphoinositidase C isoforms are specifically localized in the nuclear matrix and cytoskeleton of Swiss 3T3 cells.

The fine subcellular localization of different phosphoinositidase C (PIC) isoforms has been determined by both electron microscope immunocytochemistry and immunoblotting in whole Swiss 3T3 cells as well as in subcellular fractions. PIC-beta, whose signaling activity has been recently demonstrated at the nuclear level (M. A. Martelli, R. S. Gilmour, V. Bertagnolo, L. M. Neri, L. Manzoli, and L. Cocco, Nature, 358, 242-244, 1992), is mainly localized in the interchromatin domains, while it is almost absent from the cytoplasm. PIC-gamma is almost absent from the nucleus and resides in the cytosol, while PIC-delta is undetectable in these cells. PIC-beta is retained in the inner nuclear matrix and lacks the nuclear pore-lamina complex, whereas PIC-gamma is preferentially associated with cytoskeletal filaments. Moreover, PIC-beta is present at the same sites of the nuclear matrix where phospholipids and protein kinase C can be identified. This indicates that some elements of the phosphoinositide signal transduction system are located inside the nucleus. Moreover, PIC-gamma association with the cytoskeleton filaments suggests a possible involvement of this enzyme in cytoskeleton-mediated changes of cell shape.

3T3 Cells↗

Immunocytochemical analysis of phosphatidylinositol-specific phospholipase C in PC12 cells: predominance of the delta isoform during neural differentiation.

The rat pheochromocytoma PC12 cell line, which differentiates into sympathetic neurons under nerve growth factor (NGF) treatment, contains at least three phosphoinositidase C (PIC) isozymes, PIC beta, PIC gamma, PIC delta. These isozymes have been previously shown to display a different subcellular localization. To determine whether or not NGF induces changes in the presence and/or distribution of PIC isozymes during PC12 neural differentiation, studies were carried out by means of in situ immunocytochemistry. After NGF administration the proliferative activity was progressively reduced to very low levels, as measured by bromodeoxy Uridine incorporation, and a neuron-like morphology was displayed by almost all cells. In unstimulated PC12 cells, PIC beta was detected in the nucleus whereas PIC delta was only cytoplasmic; PIC gamma was found in both cell compartments. In cells treated with NGF for 3 days, neural processes extended to twice the diameter of the cell body; the gamma isoform was concentrated near the nucleus, while the immunoreactivity of the beta form remained constant and the delta form was increased. After 10 days of treatment with NGF, PIC beta was hardly detectable and PIC gamma immunostaining was considerably decreased. On the contrary, PIC delta progressively increased and, after 14 days of NGF exposure, fully differentiated cells displayed an intense labelling of cell body and neurites. In the same cells, PIC beta and PIC gamma were almost negative. These results suggest that NGF dependent neural differentiation is related to the selective down regulation of PIC beta and gamma and the increase of PIC delta isozyme associated with the decrease of cell proliferation.

Animals↗

Nuclear phosphoinositidase C during growth factor stimulation.

The existence of phosphoinositidase C beta is demonstrated in the nucleus of Swiss 3T3 cells. Moreover, we show that this isoform is specific for the nucleus while the gamma isoform is confined to the cytoplasm of these cells. When Swiss 3T3 fibroblasts are treated with Insulin-like Growth Factor I a rapid and transient activation of the beta isoform occurs at the nucleus whilst the cytoplasmic phosphoinositidase C is unaffected. These results seem to explain the mechanism by which the decrease in the mass of polyphosphoinositol lipids occurs in the nucleus after stimulation with Insulin-like Growth Factor I and actually point out the existence of a nuclear polyphosphoinositide signalling system distinct from the plasma membrane localized system and constituted not only by the previously demonstrated lipid kinases but also by a specific phosphoinositidase C.

3T3 Cells↗

Discrete subcellular localization of phosphoinositidase C beta, gamma and delta in PC12 rat pheochromocytoma cells.

Phosphoinositidase C activity was revealed in nuclei isolated from PC12 rat pheochromocytoma cells incubated with tritiated phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Phosphoinositide breakdown was found to be optimal at neutral pH and Ca++ concentrations ranging from endogenous levels to millimolar values. To characterize the enzymes involved, three monoclonal antibodies directed against the beta, gamma and delta phosphoinositidase C isoforms were employed. A combination of Western blot immunochemical analysis on cytoplasmic and nuclear fractions and of in situ immunocytochemistry on intact cells and isolated nuclei indicated that phosphoinositidase C gamma, though predominantly cytoplasmic, was present in both cell compartments. On the contrary, phosphoinositidase C beta was exclusively localized in the nucleus, whereas phosphoinositidase C delta was restricted to the cytoplasm. These data suggest that inositol lipid breakdown is controlled by different phosphoinositidase C isozymes in the various cell compartments, and support the notion that a separate phosphoinositide signalling system is located in the nucleus.

Animals↗

Natural killer function in flow cytometry. III. Surface marker determination of K562-conjugated lymphocytes by dual laser flow cytometry.

The recognition of effector cell populations that are able to actively from conjugates with target cells is of major importance in studies of lymphocyte cytotoxicity. A number of methodologies have been described to identify the conjugates and count them, but there have been few studies of the binding capability of the different subsets of effector cells involved in the conjugation phenomenon. Here we describe a methodology that permits the study of two surface markers on lymphocytes conjugated to K562 target cells. In particular, the expression of low density CD8 (CD8dim) has been studied on both CD3+ and CD16+ lymphocytes bound to K562 target cells. Previously described methodologies, either optical microscopy or flow cytometry, were not able to identify the effector population by mAb double staining, especially in the case of antigens expressed at low density. The flow cytometric methodology described here permits the measurement of the binding activity of small lymphocyte subsets such as the CD3+ 8dim+ population. However, the method could be used to study the binding activity of any effector population defined by mAb double staining.

Antigens, Differentiation↗

Morphological evidence of function-related localization of phospholipids in the cell nucleus.

The evidence accumulated in recent years on the presence of phospholipids inside the interphase nucleus needs a precise localization of the nuclear sites of accumulation, transport and degradation of these molecules. A very useful approach for monitoring the fine localization of nuclear phospholipids is represented by a recently developed technique using gold-conjugated phospholipases. In fact, in addition to the phospholipids organized in bilayers in the membrane, this technique identifies amorphous lipoprotein complexes present in different cell areas as well as in the nucleus. In this way and using sample preparation systems which reduce lipid removal and translocation, such as cryofixation, cryosectioning, embedding in hydrophylic resins and cryofracturing, we have analyzed the subnuclear localization of phospholipids in different experimental conditions. The results indicate that: in interphase the nuclear phospholipids are localized mainly in the interchromatin spaces and in the nucleolar domain; the observed co-localization of phospholipids and ribonucleoproteins suggests that phospholipids are involved in the mechanism of transport and release of the transcripts; the demonstrated release of ribonucleoproteins after phospholipase digestion suggests that phospholipids mediate the binding between ribonucleoproteins and the nuclear matrix; significant changes of the phospholipid localization occur in the different phases of the cell cycle or in the course of induced cell differentiation.

Animals↗