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Monocytic differentiation of HL-60 cells is characterized by the nuclear translocation of phosphatidylinositol 3-kinase and of definite phosphatidylinositol-specific phospholipase C isoforms.
No full textImmunochemical and immunocytochemical data indicate that nuclei of HL-60 cells contain different enzymes involved in the phosphoinositide cycle, such as PI 3-K and the phosphatidylinositol-specific PLC isoforms beta3, gamma1 and gamma2. These enzymes translocate differently to the nuclear fraction when HL-60 cells are treated with differentiating doses of vitamin D3: PI 3-K translocated progressively to the nucleus in parallel with full differentiation until 96 hours. PLC beta3 increased until 72 hours of treatment and then lowered its intranuclear amount and PLC gamma1 was unchanged at all the examined times. PLC gamma2 nuclear translocation increased progressively until 96 hours of vitamin D3 administration. A fourth PLC isozyme, beta2, present in the cytoplasm of untreated cells, translocates to the cytoplasm after vitamin D3 addition and reaches the highest concentration at the end of monocytic differentiation. Terminal monocytic differentiation was characterized at the nuclear level by high levels of PI 3-K and PLC gamma2 and by the novel expression of PLC beta2. We then observed that the xi isoform of PKC, constitutively present in nuclei of HL-60 cells, translocated to the nucleus when cells were induced to differentiate along the monocytic lineage, but the nuclear translocation of PKC xi was blocked as a consequence of PI 3-K inhibition by Wortmannin. These findings indicate that the main components of the noncanonical and canonical inositol lipid signal transduction pathways, including PI 3-K, PLC beta2 and beta3, PLC gamma2, undergo nuclear translocation and may therefore play a relevant role during monocytic differentiation at the nuclear level. Furthermore, PKC xi nuclear translocation appears to be related to PI 3-K activity
Phosphatidylinositol 3-kinase in HL-60 nuclei is bound to the nuclear matrix and increases during granulocytic differentiation.
No full textWe 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
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.
No full textWe 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
The controversial role of cAMP on amnionic prostaglandin release: Effect of adenylate cyclase inhibition
No full textThe suggested role of cAMP in the regulation of amnionic prostaglandin release was investigated using two adenylate cyclase inhibitors, MDL 12330A and SQ 22536. These substances exhibited a dose-dependent inhibitory effect on both amnionic enzyme and cAMP levels, but they did not influence prostaglandin E (PGE) release. In addition forskolin and IBMX (3-isobutyl-1-methylxanthine), two drugs known to increase cAMP levels, did not affect PGE output, while dibutyryl cyclic cAMP showed a dose-dependent inhibitory effect. On the basis of our data, the suggested role of amnionic adenylate cyclase in triggering prostaglandin release is not confirmed, and the pathway of phospholipase A 2 activation at the onset of labor remains to be elucidated
Selective up-regulation of phospholipase C-beta 2 during granulocytic differentiation of normal and leukemic hematopoietic progenitors
No full textIn this study, we have investigated the expression of phospholipase C-beta2 during the course of granulocytic differentiation of normal and malignant progenitors. As a model system, we used the NB4 cell line, a reliable in vitro model for the study of acute promyelocytic leukemia (APL), a variety of acute myeloid leukemia (AML) that responds to pharmacological doses of all trans-retinoic acid (ATRA) by differentiating in a neutrophil-like manner. We found that PLC-beta2, virtually absent in untreated NB4 cells, was strongly up-regulated after ATRA-induced granulocytic differentiation. Remarkably, using primary blasts purified from bone marrow of patients affected by APL successfully induced to remission by treatment with ATRA, we showed a striking correlation between the amount of PLC-beta2 expression and the responsiveness of APL blasts to the differentiative activity of ATRA. An increase of PLC-beta2 expression also characterized the cytokine-induced granulocytic differentiation of CD34+ normal hematopoietic progenitors. Taken together, these data show that PLC-beta2 represents a sensitive and reliable marker of neutrophil maturation of normal and malignant myeloid progenitors. Moreover, PLC-beta2 levels can predict the in vivo responsiveness to ATRA of APL patients
Phosphatidylinositol 3-kinase in HL-60 nuclei is bound to the nuclear matrix and increases during granulocytic differentiation
No full textWe 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
Requirement of tyrosine-phosphorylated Vav for morphological differentiation of all-trans-retinoic acid-treated HL-60 cells.
No full textOur previous data demonstrated that cellular and nuclear tyrosine-phosphorylated Vav associate with phosphoinositide 3-kinase during all-trans-retinoic acid-dependent granulocytic differentiation of HL-60 cells. In this study, aimed to analyze the mechanism by which Vav is recruited and activated, we report that the Src homology 2 domain of Vav interacts with tyrosine-phosphorylated proteins in a differentiation-dependent manner. Two adaptor proteins, Cbl and SLP-76, were identified, showing a discrete distribution inside the cells, with Cbl absent from the nuclei and SLP-76 particularly abundant in the nuclear compartment. Of note, Vav interacts with the tyrosine kinase Syk, which is also present in the nuclear compartment and may phosphorylate Vav in vitro when cells differentiate. Inhibition of Syk activity by piceatannol prevents both in vitro and in vivo Vav tyrosine phosphorylation, its association with the regulatory subunit of phosphoinositide 3-kinase, and the nuclear modifications typically observed during granulocytic differentiation of this cell line. These findings suggest that tyrosine-phosphorylated Vav and its association with phosphoinositide 3-kinase play a crucial role in all-trans-retinoic acid-induced reorganization of the nucleoskeleton, which is responsible for the changes in nuclear morphology observed during granulocytic differentiation of HL-60 cells
Stromal derived factor-1 alpha (SDF-1 alpha) induces CD4+ T cell apoptosis via the functional up-regulation of the Fas (CD95)/Fas ligand (CD95L) pathway.
No full textStromal-derived factor-1alpha (SDF-1alpha), the high-affinity ligand of CXC-chemokine receptor 4 (CXCR4), induced a progressive increase of apoptosis when added to the Jurkat CD4+/CXCR4+ T cell line. The SDF-1alpha-mediated Jurkat cell apoptosis was observed in serum-free or serum-containing cultures, peaked at SDF-1alpha concentrations of 10-100 ng/ml, required 3 days to take place, and was completely blocked by the z-VAD-fmk tripeptide caspase inhibitor. Although SDF-1alpha did not modify the expression of TNF-alpha or that of TNF-RI and TNF-RII, it increased the expression of surface Fas/APO-1 (CD95) and intracellular Fas ligand (CD95L) significantly. Moreover, the ability of SDF-1alpha to induce apoptosis was inhibited by an anti-CD95 Fab' neutralizing antibody. These findings suggest a role for SDF-1alpha in the homeostatic control of CD4+ T-cell survival/apoptosis mediated by the CD95-CD95L pathway
Selective up-regulation of phospholipase C-beta2 during granulocytic differentiation of normal and leukemic hematopoietic progenitors.
No full textIn this study, we have investigated the
expression of phospholipase C-2 during the
course of granulocytic differentiation of normal
and malignant progenitors. As a model system, we
used the NB4 cell line, a reliable in vitro model for
the study of acute promyelocytic leukemia (APL),
a variety of acute myeloid leukemia (AML) that
responds to pharmacological doses of all transretinoic
acid (ATRA) by differentiating in a neutrophil-
like manner. We found that PLC-2, virtually
absent in untreated NB4 cells, was strongly upregulated
after ATRA-induced granulocytic differentiation.
Remarkably, using primary blasts purified
from bone marrow of patients affected by APL
successfully induced to remission by treatment with
ATRA, we showed a striking correlation between
the amount of PLC-2 expression and the responsiveness
of APL blasts to the differentiative activity
of ATRA. An increase of PLC-2 expression also
characterized the cytokine-induced granulocytic
differentiation of CD34 normal hematopoietic
progenitors. Taken together, these data show that
PLC-2 represents a sensitive and reliable marker
of neutrophil maturation of normal and malignant
myeloid progenitors. Moreover, PLC-2 levels can
predict the in vivo responsiveness to ATRA of APL
patients
Retinoic acid maintains differentiated cell morphology and functions in long-term cultured porcine hepatocytes: Obtaining functional cells for prolonged treatments with bioartificial liver
No full textPorcine hepatocytes show several immunological characteristics and enzymatic activities of human liver, representing an ideal xenogenic source of cells as biological component of bioartificial liver (BAL). Isolated hepatocytes rapidly lose their specific metabolic activities and their typical morphology when cultured in the presence of serum. Since in BAL porcine hepatocytes are perfused by the patient's plasma, procedures able to minimize de-differentiation of cells could be useful for long-term treatment of acute liver failure (ALF). In this work we found that, in the presence of micromolar concentration of All trans-retinoic acid (ATRA), porcine parenchymal liver cells undergo to a lower extent the de-differentiating effects of long-term culture in the presence of serum. The evaluation of lidocaine metabolism showed that ATRA-treated cells retain specific hepatocyte function for a significantly longer time when compared to control hepatocytes. A tyrosine phosphorylation of PLC-gamma1 was observed in concomitance with the ATRA-induced maximal functional activity. An increased expression of PLC-beta3 and PKC-alpha and -beta2 was also evidentiated at the longer time points explored, when the effects of ATRA in preservation of the differentiated morphology were maximal. These results provide the first evidence that ATRA plays a differentiating role in adult porcine hepatocytes cultured under de-differentiating conditions. The administration of ATRA to isolated parenchymal cells from pig liver may provide functional hepatocytes for prolonged treatment with BAL
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