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L Cocco

Publications and source records attributed to L Cocco.

At least 73 records · Page 4Linked to original sources
PubMed1995

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins.

Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at 0 degrees C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37 degrees C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37 degrees C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37 degrees C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37 degrees C in vitro, unlike to that what previous reports have indicated.

Animals↗
PubMed1995

Inositides in nuclei of Friend cells: changes of polyphosphoinositide and diacylglycerol levels accompany cell differentiation.

Friend erythroleukemia cells were labelled with high levels of [3H]myo-inositol and the radioactivity in PI, PIP and PIP2, extracted from isolated nuclei, was measured. A parallel analysis employing a picomole sensitive assay for both PIP and DAG has been carried out. The results indicate that the differentiation process is characterised by an accumulation of nuclear PIP and PIP2 and by a decrease of DAG mass. We suggest that as differentiation proceeds toward erythrocytes in Friend cells, this is accompanied by a reduction in the amount of these messengers in the nucleus.

Animals↗
PubMed1995

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↗
PubMed1994

Selective nuclear translocation of protein kinase C alpha in Swiss 3T3 cells treated with IGF-I, PDGF and EGF.

To determine the subcellular distribution of PKC after GFs treatment we have employed a combined immunochemical and in situ confocal microscopy analysis. In quiescent Swiss 3T3 cells only a faint PKC positivity was observable in the nucleus while a strong reaction was seen in the cytoplasm. IGF-I and to a lesser extent PDGF and EGF induced, after 45 min of treatment, a nuclear translocation of PKC detected by a pan-anti-PKC antibody and nuclear fluorescence was distributed in the nuclear interior except for the nucleolar regions. Bombesin and FGF did not affect the sub-cellular distribution of the enzyme. To further the understanding of which PKC isoform was involved in the translocation process, we have tested nine isozyme-specific anti-PKC antibodies. Immunoblotting analysis revealed the presence in Swiss 3T3 fibroblasts of alpha, beta I, epsilon and zeta isoforms. In isolated nuclei from GF-exposed cells only the alpha isozyme was detected: immunostaining was very intense after IGF-I treatment and clearly observable after PDGF and EGF stimulation. This result was strongly supported by the in situ confocal microscopy which parallels the Western blot analysis. These data demonstrate that several, but not all, GFs acting through tyrosine kinase receptor induce the intranuclear translocation of PKC alpha and, because of the dramatic effect of IGF-I, strengthen the case for a link between the activation of nuclear inositol lipid cycle and PKC translocation induced by this GF.

3T3 Cells↗
PubMed1994

Phosphoinositide signaling in nuclei of Friend cells: phospholipase C beta down-regulation is related to cell differentiation.

Previous investigations have demonstrated the existence of an autonomous intranuclear inositide cycle endowed with conventional lipid kinases and phospholipase C (PLC) which is the isoform beta in Swiss 3T3 cells, PC12 pheochromocytoma cells, human osteosarcoma SaOS-2 cells, and rat liver. The presence of PLC has been investigated in nuclei of Friend erythroleukemia cells. Both beta and gamma isoforms are present in these nuclei. When Friend cells undergo terminal erythroid differentiation in the presence of dimethyl sulfoxide the PLC beta isoform is down-regulated as shown by immunochemical and immunocytochemical analysis, by determination of enzymatic activity directly and in the presence of neutralizing monoclonal antibodies and also by Northern blot for PLC beta message. By contrast, the amount of PLC gamma and its activity are unaffected by erythroid differentiation. Thus, the presence of a nuclear PLC beta, the activity and expression of which are modulated during differentiation of erythroleukemia cells, implicates a role for nuclear phosphoinositide signaling in the processes of cell determination and indicates the nuclear PLC beta as a key enzyme of the cycle in relation to the erythroid differentiative commitment of murine erythroleukemia cells.

Animals↗
PubMed1994

Interleukin 1 alpha stimulates nuclear phospholipase C in human osteosarcoma SaOS-2 cells.

Interleukin 1 (IL-1) is one of the most potent stimulators of bone resorption. However, the early biochemical events elicited by IL-1 receptor binding are not fully understood. Here we show that in human osteosarcoma SaOS-2 cells the treatment with IL-1 alpha is able to evoke a rapid and transient increase of nuclear phospholipase C (PLC) activity. A parallel decrease of nuclear phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate is observed. All these events are strictly confined to the nuclear compartment without affecting the cytoplasmatic inositol lipid pool. In addition we show that by Western blot analysis with specific monoclonal antibodies the PLC gamma is located both in the cytoplasm and in the nucleus, while PLC beta appears exclusively localized in the nucleus. Moreover, the increase of PLC activity in response to IL-1 alpha is completely neutralized by monoclonal antibody against the beta-form. While confirming the existence of an autonomous nuclear phosphoinositide signaling system, our data clearly indicate that in SaOS-2 cells one of the earliest events following IL-1 alpha treatment is the breakdown of nuclear phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate because of the activation of a specific nuclear PLC isoform.

Cell Nucleus↗
PubMed1994

No discrete complexes containing DNA polymerase alpha activity can be solubilized from the heat-stabilized nuclear matrix prepared from HeLa S3 cells.

Most of the DNA polymerase alpha activity, bound to the heat-stabilized nuclear matrix prepared from HeLa S3 cells, was released as a matrix extract by sonication. When the extract was centrifuged in a 5-20 per cent linear sucrose gradient no definite peaks of activity could be identified. Most of the activity sedimented to the bottom of the tube under all the conditions tested, whilst the remaining activity was associated with matrix fragments of various and irregular size. No 10 S complexes, containing polymerase activity, were seen after incubation of the extract for 16 h before centrifugation. Other solubilization procedures (i.e. treatment of the matrix with chelating agents, high pH associated with reducing agents, ionic and nonionic detergents) failed to produce release of matrix-bound DNA polymerase alpha activity. In contrast, we released 10 S complexes, containing polymerase activity, from the matrix prepared from nuclei not exposed to heat. We conclude that a 37 degrees C incubation of isolated nuclei before extraction with 2 M NaCl and DNase I digestion causes DNA polymerase alpha to bind to the nuclear matrix in a form that cannot subsequently be released as discrete components, at variance with previous results obtained with the matrix prepared from regenerating rat liver.

Centrifugation, Density Gradient↗
PubMed1994

6-Iodoacetamidofluorescein labelling to assess the state of sulphhydril groups after thermal stabilization of isolated nuclei.

Isolated nuclei and nuclear matrices, prepared from mouse erythroleukaemia cells, were reacted with the sulphhydryl-specific dye 6-iodoacetamidofluorescein. To determine whether in vitro formation of disulphide bonds might play a role in the nuclear matrix stabilization triggered by exposure of isolated nuclei to the physiological temperature of 37 degrees C, a variety of techniques were employed to assess the state of cysteinyl residues after such an incubation. Both flow cytometry and confocal microscopy quantitative analysis did not reveal major differences in the fluorescence intensity of nuclei incubated at 37 degrees C in comparison with those maintained at 0 degrees C. Confocal scanning laser microscopy revealed that 6-iodoacetamidofluorescein labelled a fibrogranular network in isolated nuclei. The fluorescent pattern of the network was not affected by a 37 degrees C exposure of nuclei. However, such a network was not detectable in isolated nuclear matrices, thus suggesting a possible protein re-arrangement during matrix preparation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of fluorescent-labelled nuclear proteins showed no difference between heat-exposed and control samples. We conclude that oxidation of cysteinyl residues is not a major factor leading to the stabilization of nuclei incubated at 37 degrees C.

Animals↗
PubMed1994

An immunohistochemical study of protein kinase C distribution in fetal mouse vertebral column.

Using polyclonal antibodies we have studied the distribution of protein kinase C in fetal mouse low thoracic vertebrae. By means of a pan protein kinase C antiserum recognizing the catalytic domain of the enzyme, we show that protein kinase C is markedly expressed in chondrocytes before birth. The enzyme seems to be very abundant in the more mature cells that are close to ossification centres as well as the periphery of the intervertebral disc, although it can also be detected in chondrocytes. In order to establish which protein kinase C isoenzyme(s) the chondrocytes produce, we employed polyclonal isoenzyme-specific antisera developed against three calcium-dependent isoforms (alpha, beta, gamma) and three calcium-independent isoforms (delta, epsilon, zeta). Secondary antibody conjugated to alkaline phosphatase revealed that chondrocytes markedly express the beta-isoform. Cells were also weakly stained by the anti-epsilon serum. The immunostaining was completely abolished by pre-incubating primary antibodies with the peptide antigens to which they were raised. These results suggest that protein kinase C (and particularly the beta isoform) could play an important role in mouse fetal chondrogenesis of the vertebral column.

Alkaline Phosphatase↗
PubMed1994

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↗
PubMed1993

Tiazofurin-induced changes in inositol lipid cycle in nuclei of Friend erythroleukemia cells.

When murine erythroleukemia cells are treated up to 96 hrs with the antitumor drug tiazofurin the induction of erythroid differentiation is accompanied by changes in both synthesis and breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5 bisphosphate in the nucleus. These changes, which parallel the effect of dimethylsulfoxide, a well known inducer of erythroid differentiation in vitro, are due to the inhibition of the nuclear phosphoinositidase C activity. Therefore the present data indicate that nuclear inositol lipid cycle as a target of tiazofurin in addition to its inhibition of IMP dehydrogenase (EC 1.1.1.205) activity.

Animals↗
PubMed1993

Age-related events in human active T lymphocytes: changes in the phosphoinositidase C activity.

Since PHA-stimulated active T lymphocytes from aged humans showed changes in the metabolic pattern of inositol lipids in comparison with young subjects, we studied the possible role of phosphoinositidase C (PIC) in the generation of this phenomenon. The breakdown of exogenous [3H]phosphatidylinositol 4,5-bisphosphate was found to be optimal at neutral pH and Ca++ concentrations close to millimolar levels. Under these conditions PIC activity of resting lymphocytes did not differ in aged and young subjects, while, after short periods of PHA stimulation (up to 4 hr) the substrate hydrolysis was lower and delayed in the elderly group in comparison with that of controls. Our findings support the hypothesis that the age-related default of this enzyme, responsible for the age-related changes in the inositol lipid pathway of this peculiar subpopulation, could be involved, as a primary event, in the mechanisms leading to the reduced proliferative response of aged active T lymphocytes.

Adult↗
PubMed1993

The protein kinase inhibitor staurosporine induces morphological changes typical of apoptosis in MOLT-4 cells without concomitant DNA fragmentation.

The protein kinase inhibitor staurosporine induces ultrastructural changes typical of apoptotic cell death in MOLT-4 cells in a concentration range of 10-200 nM. The well known chromatin margination was indeed present, followed by characteristic nuclear protrusions. The formation of numerous homogeneously electron dense micronuclei was the final step of the process. Nevertheless we did not detect the distinctive internucleosomal DNA fragmentation which has been demonstrated to occur in a variety of cells exposed to agents causing apoptosis. Our results strengthen recent argument showing that DNA breakage cannot be considered the sole criterion for detection of apoptosis.

Alkaloids↗
PubMed1993

Absence of high levels of DNA polymerase alpha activity in the nuclear matrix prepared from mouse erythroleukemia cells.

We have examined the association of DNA polymerase alpha activity with the nuclear matrix prepared by different techniques from mouse erythroleukemia cells. At variance with the data obtained using other cell types we have found that only a small amount (less than 2%) of nuclear DNA polymerase alpha activity resisted extraction with high-ionic strength buffers, even if nuclei were heat-stabilized by incubation at 37 degrees C for 45 min prior to subfractionation. The recovery of DNA polymerase alpha activity bound to the matrix was unaffected by the type of extracting agent used (NaCl or (NH4)2 SO4), by the extraction sequence or by the method employed for obtaining nuclei. These results could indicate that in some types of cells the nuclear matrix is not involved in DNA replication.

Animals↗
PubMed1993

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↗
PubMed1993

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↗
PubMed1992

Nuclear localization and signalling activity of phosphoinositidase C beta in Swiss 3T3 cells.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is a widespread receptor-coupled signalling system at the plasma membrane of most eukaryotic cells. The existence of an entirely separate nuclear phosphoinositide signalling system is suggested from evidence that purified nuclei synthesize PtdInsP2 and phosphatidylinositol 4-phosphate (PtdInsP) in vitro and that a transient decrease in the mass of these lipids occurs when Swiss 3T3 cells are cultured in the presence of insulin-like growth factor-1 (IGF-1). These IGF-1-dependent changes in inositol lipids coincide with an increase in nuclear diacyglycerol and precede translocation to the nucleus and activation of protein kinase C (refs 5, 6). Circumstantial evidence that links these changes with mitosis comes from the isolation of a 3T3 clone that expresses the type-1 IGF receptor and binds IGF-1 peptide but does not respond mitogenically or show transient mass changes in nuclear inositol lipids. A key question is how IGF-1 initiates the rapid breakdown of PtdInsP and PtdInsP2 in the nucleus. Here we present evidence that nuclei of 3T3 cells contain the beta-isozyme of phosphoinositidase C, whereas the gamma-isozyme is confined to the cytoplasm and that IGF-1 treatment stimulates exclusively the activity of nuclear phosphoinositidase C.

3T3 Cells↗