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S Capitani

Publications and source records attributed to S Capitani.

At least 55 records · Page 3Linked to original sources
PubMed1999

Influence of different metal ions on the ultrastructure, biochemical properties, and protein localization of the K562 cell nuclear matrix.

The higher order of chromatin organization is thought to be determined by the nuclear matrix, a mainly proteinaceous structure that would act as a nucleoskeleton. The matrix is obtained from isolated nuclei by a series of extraction steps involving the use of high salt and nonspecific nucleases, which remove chromatin and other loosely bound components. It is currently under debate whether these structures, isolated in vitro by unphysiological extraction buffers, correspond to a nucleoskeleton existing in vivo. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions steps; rather, it must be stabilized before the application of extracting agents. In this study nuclei, isolated from K562 human erythroleukemia cells, were stabilized by incubation with different metal ions (Ca2+, Cu2+, Zn2+, Cd2+), and the matrix was obtained by extraction with 2 M NaCl. By means of ultrastructural analysis of the resulting structures, we determined that, except for Ca2+, all the other metals induced a stabilization of the matrix, which retained the inner fibrogranular network and residual nucleoli. The biochemical composition, analyzed by two-dimensional gel electrophoresis separation, exhibited a distinct matrix polypeptide pattern, characteristic of each type of stabilizing ion employed. We also investigated to what extent metal ions could maintain in the final structures the original distribution of three inner matrix components, i.e. NuMA, topoisomerase IIalpha, and RNP. Confocal microscopy analysis showed that only NuMa, and, to a lesser extent, topoisomerase IIalpha, were unaffected by stabilization with divalent ions. On the contrary, the fluorescent RNP patterns detected in the resulting matrices were always disarranged, irrespective of the stabilization procedure. These results indicate that several metal ions are powerful stabilizing agents of the nuclear matrix prepared from K562 erythroleukemia cells and also strengthen the concept that NuMA and topoisomerase IIalpha may act as structural components of the nuclear matrix.

Antigens, Neoplasm↗
PubMed1999

Selective modulation of specific protein kinase C (PKC) isoforms in primary human megakaryocytic vs. erythroid cells.

We have investigated the pattern of expression of classical (alpha, betaI, betaII, gamma), novel (delta) and atypical (zeta) protein kinase C (PKC) isoforms during the course of human hematopoietic differentiation along the closely related megakaryocytic and erythroid lineages. Using in situ immunofluorescence analysis, freshly isolated human pluripotent CD34+ hematopoietic progenitor cells expressed detectable amounts of all the PKC isoforms investigated. On the other hand, clear-cut differences in terms of PKC staining were noticed between cells belonging to the erythroid and megakaryocytic lineages, obtained after 9 days of serum-free liquid culture in the presence of specific growth factors. Specifically, 1) erythroid cells showed a very weak expression of PKC-alpha, -betaI, -betaII, and -gamma, while megakaryocytes showed an enhanced expression of all classical PKC isoforms, predominantly confined to the cytoplasm; 2) the expression of PKC-delta increased in the cytoplasmic and nuclear compartments of both erythroid and megakaryocytic cells with respect to CD34+ cells; and 3) atypical PKC-zeta isoform showed a striking accumulation in the nucleus during both erythroid and megakaryocytic differentiation.

Cell Differentiation↗
PubMed1999

Lineage-restricted expression of protein kinase C isoforms in hematopoiesis.

The pattern of expression of several protein kinase C (PKC) isoforms (alpha, betaI, delta, epsilon, eta, and zeta) during the course of hematopoietic development was investigated using primary human CD34(+) hematopoietic cells and stable cell lines subcloned from the growth factor-dependent 32D murine hematopoietic cell line. Each 32D cell clone shows the phenotype and growth factor dependence characteristics of the corresponding hematopoietic lineage. Clear-cut differences were noticed between erythroid and nonerythroid lineages. (1) The functional inhibition of PKC-epsilon in primary human CD34(+) hematopoietic cells resulted in a twofold increase in the number of erythroid colonies. (2) Erythroid 32D Epo1 cells showed a lower level of bulk PKC catalytic activity, lacked the expression of epsilon and eta PKC isoforms, and showed a weak or absent upregulation of the remaining isoforms, except betaI, upon readdition of Epo to growth factor-starved cells. (3) 32D, 32D GM1, and 32D G1 cell lines with mast cell, granulo-macrophagic, and granulocytic phenotype, respectively, expressed all the PKC isoforms investigated, but showed distinct responses to growth factor readdition. (4) 32D Epo 1.1, a clone selected for interleukin-3 (IL-3) responsiveness from 32D Epo1, expressed the epsilon isoform only when cultured with IL-3. On the other hand, when cultured in Epo, 32D Epo1.1 cells lacked the expression of both epsilon and eta PKC isoforms, similarly to 32D Epo1. (5) All 32D cell lines expressed the mRNA for PKC-epsilon, indicating that the downmodulation of the epsilon isoform occurred at a posttranscriptional level. In conclusion, the PKC isoform expression during hematopoiesis appears to be lineage-specific and, at least partially, related to the growth factor response.

Animals↗
PubMed1999

Extracellular HIV-1 tat protein up-regulates the expression of surface CXC-chemokine receptor 4 in resting CD4+ T cells.

Here we report that synthetic HIV-1 Tat protein, immobilized on a solid substrate, up-regulates the surface expression of the CXC-chemokine receptor 4 (CXCR4), but not of the CC-chemokine receptor 5 in purified populations of primary resting CD4+ T cells. The Tat-mediated increase of CXCR4 occurred in a well-defined range of concentrations (1-10 nM of immobilized Tat) and time period (4-8 h postincubation). Moreover, the increase of CXCR4 was accompanied by an increased entry of the HXB2 T cell line-tropic (X4-tropic), but not of the BaL macrophage-tropic strain of HIV-1. The ability of Tat to up-regulate CXCR4 expression was abrogated by the protein synthesis inhibitor cycloheximide, clearly indicating the requirement of de novo synthesis. As Tat protein is actively released by HIV-1 infected cells, our data indicate a potentially important role for extracellular Tat in rendering bystander CD4+ T cells more susceptible to infection with X4-tropic HIV-1 isolates.

Adult↗
PubMed1999

Human immunodeficiency virus type 1 Nef protein sensitizes CD4(+) T lymphoid cells to apoptosis via functional upregulation of the CD95/CD95 ligand pathway.

Many viruses have evolved genes encoding proteins that regulate cell death by apoptosis. The human immunodeficiency virus type 1 (HIV-1) Nef protein alters T-cell development and signaling and is required for optimal viral replication and pathogenicity in vivo. To analyze the interference of Nef with cell survival, we used both regulated and constitutively expressed nef alleles in stably transfected T-cell lines. Nef-expressing cells were sensitized to cell death by apoptosis, which was specifically exacerbated by an anti-CD95 IgM monoclonal antibody (MoAb). Flow cytometric analysis showed that the surface expression of both CD95 and CD95 ligand (CD95L) was upregulated by endogenous Nef expression. Nef-mediated apoptosis was almost completely suppressed by the addition in culture of an anti-CD95 Fab' IgG MoAb, which specifically blocks CD95/CD95L interactions. Lastly, mutation of a proline motif in the core region of the nef gene, which disrupts its ability to interact with cellular kinases and reduces HIV-1 replication in vitro, completely abrogated the Nef-mediated induction of apoptosis as well as its ability to upregulate surface CD95 and CD95L. These findings may provide molecular insight into the role of endogenous Nef in the T-cell depletion observed in vivo, particularly HIV-specific cytotoxic CD8(+) T cells.

Apoptosis↗
PubMed1999

Phosphoinositide 3-kinase activity is essential for all-trans-retinoic acid-induced granulocytic differentiation of HL-60 cells.

Phosphoinositide 3-kinase (PI 3-K) activity increases in HL-60 cells that are induced to granulocytic differentiation by all-trans-retinoic acid. Immunochemical and immunocytochemical analyses by confocal microscopy also reveal an increase in the amount of the enzyme, which is particularly evident at the nuclear level. Inhibition of PI 3-K activity by nanomolar concentrations of wortmannin and of its expression by transfection with an antisense fragment of p85alpha prevented the differentiative process. The data obtained indicate that PI 3-K activity plays an essential role in promoting granulocytic differentiation.

Antineoplastic Agents↗
PubMed1999

Lamin A is part of the internal nucleoskeleton of human erythroleukemia cells.

Nuclear lamins are the most abundant components of the nuclear lamina, a 10-50-nm-thick fibrous layer underlying the inner nuclear envelope membrane. Nevertheless, a number of recent investigations performed on epithelial and fibroblast cells have suggested that nuclear lamins are also present within the nucleoplasm and could be important constituents of the nucleoskeleton. We have studied the subnuclear distribution of lamins A and B1 in human erythroleukemia cells by using immunoblotting analysis and immunofluorescent staining of fractionated nuclei. In intact cells and isolated nuclei, antibodies to lamins A and B1 mainly stained the nuclear periphery, although some immunoreactivity was detected in the nuclear interior. However, when chromatin was removed by nuclease digestion and extraction with nonionic detergent or solutions of high ionic strength, a previously masked immunoreactivity for lamin A, but not for lamin B1, became evident in the internal part of the residual structures representing the nuclear matrix or scaffold. Preferential localization of lamin A to the inner part of the nucleus was also demonstrated by the presence of the majority of lamin A in the solubilized inner nuclear network subfraction. In contrast, lamin B1 was mainly recovered in the fraction corresponding to the nuclear periphery. Double labeling experiments showed that lamin A, but not lamin B1, colocalized with coiled and GATA-1 bodies. Thus, our results support the hypothesis that lamin A, but not lamin B1, may be a component of an internal nucleoskeleton in human erythroleukemia cells.

Cell Cycle↗
PubMed1999

Inositides in the nucleus: further developments on phospholipase C beta 1 signalling during erythroid differentiation and IGF-I induced mitogenesis.

Inositol lipids originally shown to be metabolized in the cytosol have been detected also in the nucleus, where they are both synthesized and hydrolyzed. In the case of erythroid differentiation of murine erythroleukemia cells (Friend cells) it has been previously shown that PLC beta 1, which is the major nuclear PLC, undergoes down-regulation upon treatment with DMSO or tiazofurin which act as differentiative agents. On the contrary, i.e., during IGF-I induced mitogenesis, it has been shown that PLC beta 1 is rapidly activated and this event is essential for the onset of DNA synthesis. Even though its key role in cell growth has been shown, both the mechanism by which nuclear PLC beta 1 is activated and the direct relationship with erythroid differentiation are still unknown. We have addressed the question if PLC beta 1 expression and activity in the nucleus are directly related or not to the establishment of the differentiated state and we have checked the two main ways of activation, i.e., via G-protein or via phosphorylation, in order to establish whether nuclear PLC beta 1 is regulated the same way as the one at the plasma membrane or not. The data reported here show that nuclear PLC beta 1 is responsible for a continuous recycling of Friend cells, acting as a negative regulator of differentiation and that its activation is dependent on the phosphorylation state.

3T3 Cells↗
PubMed1999

Selective modulation of the cyclin B/CDK1 and cyclin D/CDK4 complexes during in vitro human megakaryocyte development.

Mammalian megakaryocyte development is characterized by a progressive accumulation of cells exhibiting a polylobated nucleus with a polyploid DNA content. In this study human megakaryocytes were obtained from CD34+ haemopoietic progenitors by in vitro liquid culture in the presence of 100 ng/ml of recombinant thrombopoietin (TPO). Ultrastructural examination of polyploid megakaryocytes showed the presence of a large number of centrioles, the breakdown of the nuclear envelope, and the progressive chromatin condensation, all aspects characteristic of mitosis. At both indirect immunofluorescence and Western blot analyses, cyclin B and its related cyclin-dependent kinase (CDK)1, which forms the mitosis promoting factor (MPF), showed an increased expression in maturating megakaryoblasts and megakaryocytes (day 8 of culture) with respect to freshly isolated CD34+ progenitors. This expression tended to decline in fully developed megakaryocytes (day 15 of culture). The amount of cyclin D and of the related CDK4, governing the G1 phase of the cell cycle, increased during megakaryocyte development, maintaining high levels of expression also in mature megakaryocytes. These results indicate that megakaryocyte polyploidization depends on a true, although incomplete, mitotic process, and that cyclin D/CDK4 probably plays a crucial role throughout megakaryocytopoiesis.

CDC2 Protein Kinase↗
PubMed1999

Low folate levels and thermolabile methylenetetrahydrofolate reductase as primary determinant of mild hyperhomocystinemia in normal and thromboembolic subjects.

Several studies have indicated that mild to moderate hyperhomocystinemia is a common cause of arterial occlusive disease. Whether hyperhomocystinemia per se is an independent risk factor for vein thromboembolism (VTE) is still somewhat controversial. Both genetic and nutritional factors influence plasma homocysteine levels. Therefore, we evaluated plasma total homocysteine (tHcy), folate, and vitamin B12 levels and established, by polymerase chain reaction, the presence of the C677T mutation (A223V) in the methylenetetrahydrofolate reductase (MTHFR) gene in 220 cases with VTE without well-established prothrombotic defects. As a control group, 220 healthy subjects from the same geographic area as the cases were investigated. Hyperhomocystinemia was defined as a plasma tHcy level above the 95th percentile in the controls (18.05 micromol/L). Hyperhomocystinemia was found in 16% of cases (odds ratio=3.59; P<0.001); deficiencies of folate (<2.47 ng/mL) or vitamin B12 (<165 pg/mL), defined as values below the 5th percentile in controls, were found in 17.7% (P<0.001) and 12.3% (P=0.015) of cases, respectively. The homozygous condition for the MTHFR mutation (VV) was present in 28.2% of cases and 17.7% of controls (odds ratio=1.82; P=0.013). Comparing only the idiopathic forms of VTE (n=80/220; 36.3%) with normal controls, individuals with hyperhomocystinemia, or individuals homozygous for MTHFR mutation increased the odds ratios to 4.03 (P=0.005) and 2.11 (P=0.018), respectively. No statistically significant difference was observed in the MTHFR genotype distribution of cases and controls with hyperhomocystinemia (P=0.386); however, the normal MTHFR genotype (AA) appeared in control subjects only when tHcy levels were below the 80th percentile (10.57 micromol/L) of the distribution, whereas in case patients, it was present at the highest tHcy levels. A strong association between mutated homozygosity (VV), low folate levels, and hyperhomocystinemia was found in both groups. We conclude that in patients with VTE who do not have coexisting prothrombotic defects, hyperhomocystinemia increases the risk of developing idiopathic and venous thrombosis; the homozygous condition for the MTHFR mutation confers a moderate risk but, together with low folate levels, it is the main determinant of mild hyperhomocystinemia in normal and thromboembolic populations.

Adult↗
PubMed1999

Lipid signaling and cell responses at the nuclear level.

The nucleus is known to be a site for an active lipid metabolism. Although phospholipids are present in the nuclear envelope, evidence suggests that they are also located further inside the nucleus. The function of these intranuclear lipids has escaped clarification for many years. Early experiments showed that they can interact with DNA double helix affecting its thermal stability and can influence RNA synthesis in isolated nuclei. However, in the last 10 years several investigations have suggested that they may be involved in signal transduction pathways at the nuclear level and a growing body of evidence supports this hypothesis.

Animals↗
PubMed1999

Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4,5) trisphosphate synthesis precede PKC-zeta translocation to the nucleus of NGF-treated PC12 cells.

We and others have previously demonstrated the existence of an autonomous nuclear polyphosphoinositide cycle that generates second messengers such as diacylglycerol (DAG), capable of attracting to the nucleus specific protein kinase C (PKC) isoforms (Neri et al. (1998) J. Biol. Chem. 273, 29738-29744). Recently, however, nuclei have also been shown to contain the enzymes responsible for the synthesis of the non-canonical 3-phosphorylated inositides. To clarify a possible role of this peculiar class of inositol lipids we have examined the question of whether nerve growth factor (NGF) induces PKC-zeta nuclear translocation in PC12 cells and whether this translocation is dependent on nuclear phosphatidylinositol 3-kinase (PI 3-K) activity and its product, phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P(3)]. NGF increased both the amount and the enzyme activity of immunoprecipitable PI 3-K in PC12 cell nuclei. Activation of the enzyme, but not its translocation, was blocked by PI 3-K inhibitors wortmannin and LY294002. Treatment of PC12 cells for 9 min with NGF led to an increase in the nuclear levels of PtdIns(3,4,5)P(3). Maximal translocation of PKC-zeta from the cytoplasm to the nucleus (as evaluated by immunoblotting, enzyme activity, and confocal microscopy) occurred after 12 min of exposure to NGF and was completely abrogated by either wortmannin or LY294002. In contrast, these two inhibitors did not block nuclear translocation of the conventional, DAG-sensitive, PKC-alpha. On the other hand, the specific phosphatidylinositol phospholipase C inhibitor, 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine, was unable to abrogate nuclear translocation of the DAG-insensitive PKC-zeta. These data suggest that a nuclear increase in PI 3-K activity and PtdIns(3,4,5)P(3) production are necessary for the subsequent nuclear translocation of PKC-zeta. Furthermore, they point to the likelihood that PKC-zeta is a putative nuclear downstream target of PI 3-K during NGF-promoted neural differentiation.-Neri, L. M., Martelli, A. M., Borgatti, P., Colamussi, M. L., Marchisio, M., Capitani, S. Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4, 5) trisphosphate synthesis precede PKC-zeta translocation to the nucleus of NGF-treated PC12 cells.

Animals↗
PubMed1998

Nuclear association of tyrosine-phosphorylated Vav to phospholipase C-gamma1 and phosphoinositide 3-kinase during granulocytic differentiation of HL-60 cells.

The granulocytic differentiation of HL-60 cells induced by all-trans retinoic acid was accompanied by a progressive tyrosine phosphorylation of specific proteins in either cells or isolated nuclei. Among these phosphoproteins, we identified the Vav adaptor in whole cells as well as in the inner nuclear compartment, where the increase in its tyrosine phosphorylation level was more conspicuous. We also demonstrated the differentiation-dependent association of nuclear phosphorylated Vav to phospholipase C-gamma1 and to the p85 regulatory subunit of phosphoinositide 3-kinase. The role of the Vav/phospholipase C-gamma1/phosphoinositide 3-kinase phosphoprotein complexes in the nuclei of HL-60 induced to differentiate along the granulocytic lineage is discussed.

Cell Differentiation↗
PubMed1998

Phosphatidylinositol 3-kinase in HL-60 nuclei is bound to the nuclear matrix and increases during granulocytic differentiation.

We have used HL-60 leukemia cells to investigate phosphatidylinositol 3-kinase (PI 3-K) during granulocytic differentiation at the nuclear level. Nuclei of HL-60 cells showed a constitutive presence of PI 3-K that increased when cells were treated with differentiating doses of ATRA. PI 3-K was also detected tightly bound to nuclear matrices of HL-60 cells, isolated by nuclease treatment and high salt extraction. Four days of ATRA treatment induced a striking increase of nuclear matrix bound PI 3-K. In situ morphological analysis by confocal microscopy showed the translocation of PI 3-K to the nucleus and to the subnuclear fractions. PI 3-K enzymatic activity was stimulated during the granulocytic differentiation process and parallelled the increase in content of nuclei and subnuclear fractions. PI 3-K activity was recovered in nuclei also without the addition of exogenous substrates, consistent with the presence of both substrates and enzyme in the nucleus. These results indicate that specific intracellular localization of PI 3-K determines the production of different phosphoinositides in the sites of the enzyme translocation, and suggest that 3-phosphoinositide metabolism may play a specific role in the nucleus, candidating PI 3-K as a key enzyme in promoting granulocytic differentiation of HL-60 cells.

Blotting, Western↗
PubMed1998

Progressive and persistent downregulation of surface CXCR4 in CD4(+) T cells infected with human herpesvirus 7.

We have previously shown that infection of CD4(+) T lymphocytes with the T-lymphotropic human herpesvirus 7 (HHV-7) downregulates surface CD4, which represents the high-affinity receptor for HHV-7. In this study, we report that HHV-7 infection also causes a progressive loss of the surface CXC-chemokine receptor 4 (CXCR4) in CD4(+) T cells, accompanied by a reduced intracellular Ca2+ flux and chemotaxis in response to stromal cell-derived factor-1 (SDF-1), the specific CXCR4 ligand. Moreover, CXCR4 is downregulated from the surface of HHV-7-infected T cells independently of CD4. Because intracellular CXCR4 antigen and mRNA levels are unaffected in productively HHV-7-infected cells, the downregulation of CXCR4 apparently does not involve a transcritional block. Since CXCR4 functions in association with CD4 to permit entry of several human immunodeficiency virus (HIV) isolates, the potential of HHV-7 to persistently downregulate the surface expression of CXCR4 may provide novel strategies for limiting HIV infection.

CD4 Antigens↗
PubMed1998

Nuclear diacylglycerol produced by phosphoinositide-specific phospholipase C is responsible for nuclear translocation of protein kinase C-alpha.

It is well established that an independent inositide cycle is present within the nucleus, where it is involved in the control of cell proliferation and differentiation. Previous results have shown that when Swiss 3T3 cells are treated with insulin-like growth factor-I (IGF-I) a rapid and sustained increase in mass of diacylglycerol (DAG) occurs within the nuclei, accompanied by a decrease in the levels of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. However, it is unclear whether or not other lipids could contribute to this prolonged rise in DAG levels. We now report that the IGF-I-dependent increase in nuclear DAG production can be inhibited by the specific phosphatidylinositol phospholipase C inhibitor 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or by neomycin sulfate but not by the purported phosphatidylcholine-phospholipase C specific inhibitor D609 or by inhibitors of phospholipase D-mediated DAG generation. Treatment of cells with 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or neomycin sulfate inhibited translocation of protein kinase C-alpha to the nucleus. Moreover, exposure of cells to 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine, but not to D609, dramatically reduced the number of cells entering S-phase upon stimulation with IGF-I. These results suggest that the only phospholipase responsible for generation of nuclear DAG after IGF-I stimulation of 3T3 cells is PI-PLC. When this activity is inhibited, neither DAG rise is seen nor PKC-alpha translocation to the nucleus occurs. Furthermore, this PI-PLC activity appears to be essential for the G0/G1 to S-phase transition.

3T3 Cells↗