Phosphoinositide signaling in nuclei of Friend cells: tiazofurin down-regulates phospholipase C beta 1.

Previous investigations have demonstrated the presence of conventional lipid kinases and phospholipase C (PLC) activities in nuclei of Friend erythroleukemia cells. Moreover, when Friend erythroleukemia cells are treated for 96 h with the antitumor drug tiazofurin, the induction of erythroid differentiation is accompanied by changes in amounts of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate due to the inhibition of an uncharacterized nuclear PLC activity. Here, we show that the nuclear PLC beta 1 isoform is down-regulated by tiazofurin (5 microM) treatment of Friend erythroleukemia cells as shown by both Western blot and Northern blot analyses for PLC beta 1 message. This indicates that PLC beta 1 down-regulation is tightly linked with erythroid differentiation of Friend erythroleukemia cells and that the autonomous nuclear signaling via inositol lipid cycle can be controlled by the antitumor drug tiazofurin.

Nuclear but not cytoplasmic phospholipase C beta 1 inhibits differentiation of erythroleukemia cells.

A body of evidence has shown the existence of a nuclear phosphoinositide cycle in different cell types. The cycle is endowed with kinases as well as phosphatases and phospholipase C (PLC). Among the PLC isozymes, the beta family is characterized by a long COOH-terminal tail that contains a cluster of lysine residues responsible for nuclear localization. Indeed, PLC beta 1 is the major isoform that has been detected in the nucleus of several cells. This isoform is activated by insulin-like growth factor I, and when this isoform is lacking, as a result of gene ablation, the onset of DNA synthesis induced by this hormone is abolished. On the contrary, PLC beta 1 is down-regulated during the erythroid differentiation of Friend erythroleukemia cells. A key question is how PLC beta 1 signaling at the nucleus fits into the erythroid differentiation program of Friend erythroleukemia cells, and whether PLC beta 1 signaling activity is directly responsible for the maintenance of the undifferentiated state of erythroleukemia cells. Here we present evidence that nuclear PLC beta 1 but not the isoform located at the plasma membrane is directly involved in maintaining the undifferentiated state of Friend erythroleukemia cells. Indeed, when wild-type PLC beta 1 is overexpressed in these cells, differentiation in response to DMSO is inhibited in that the expression of beta-globin is almost completely abolished, whereas when a mutant lacking the ability to localize to the nucleus is expressed, the cells differentiate, and the expression of beta-globin is the same as in wild-type cells.

Identification and chromosomal localisation by fluorescence in situ hybridisation of human gene of phosphoinositide-specific phospholipase C beta(1).

Members of phosphoinositide-specific phospholipase C (PLC) families are central intermediary in signal transduction in response to the occupancy of receptors by many growth factors. Among PLC isoforms, the type beta(1) is of particular interest because of its reported nuclear localisation in addition to its presence at the plasma membrane. It has been previously shown that both the stimulation and the inhibition of the nuclear PLCbeta(1) under different stimuli implicate PLCbeta(1) as an important enzyme for mitogen-activated cell growth as well as for murine erythroleukaemia cell differentiation. The above findings hinting at a direct involvement of PLCbeta(1) in controlling the cell cycle in rodent cells, and the previously reported mapping of its gene in rat chromosome band 3q35-36, a region frequently rearranged in rat tumours induced by chemical carcinogenesis, prompted us to identify its human homologue. By screening a human foetal brain cDNA library with the rat PLCbeta(1) cDNA probe, we have identified a clone homologous to a sequence in gene bank called KIAA 0581, which encodes a large part of the human PLCbeta(1). By using this human cDNA in fluorescence in situ hybridisation on human metaphases, it has been possible to map human PLCbeta(1) on chromosome 20p12, confirming the synteny between rat chromosome 3 and human chromosome 20 and providing a novel locus of homology between bands q35-36 in rat and p12 in man. Since band 20p12 has been recently reported amplified and/or deleted in several solid tumours, the identification and chromosome mapping of human PLCbeta(1) could pave the way for further investigations on the role exerted both in normal human cells and in human tumours by PLCbeta(1), which has been shown to behave as a key signalling intermediate in the control of the cell cycle.

Inositides in the nucleus: presence and characterisation of the isozymes of phospholipase beta family in NIH 3T3 cells.

Previous reports from our laboratories and others have hinted that the nucleus is a site for an autonomous signalling system acting through the activation of the inositol lipid cycle. Among phospholipases (PLC) it has been shown previously that PLCbeta1 is specifically localised in the nucleus as well as at the plasma membrane. Using NIH 3T3 cells, it has been possible to obtain, with two purification strategies, in the presence or in the absence of Nonidet P-40, both intact nuclei still maintaining the outer membrane and nuclei completely stripped of their envelope. In these nuclei, we show that not only PLCbeta1 is present, but also PLCbeta2, PLCbeta3 and PLCbeta4. The more abounding isoform is PLCbeta1 followed by PLCbeta3, PLCbeta2 and PLCbeta4, respectively. All the isoforms are enriched in nuclear preparations free from nuclear envelope and cytoplasmatic debris, indicating that the actual localisation of the PLCbeta isozymes is in the inner nuclear compartment.

Increased phosphorylation of nuclear substrates for rat brain protein kinase C in regenerating rat liver nuclei.

Protein phosphorylation catalysed by rat brain protein kinase C (PKC) has been studied in nuclei isolated from normal and regenerating rat liver. Histone H1 and a 40,000 molecular weight protein were hyperphosphorylated at all the explored regeneration times, ranging from 3 to 22 h after partial hepatectomy. Phosphorylation of the two substrates was totally dependent on calcium and lipids and was abolished by low concentration of staurosporine. The observed early change of phosphate content of histone H1 and of the 40,000 molecular weight protein on the time scale of liver regeneration suggests that PKC might be involved in the initial nuclear events leading to cell proliferation.

Cyclosporin A specifically affects nuclear PLCbeta1 in immunodepressed heart transplant patients with gingival overgrowth.

One of the most commonly observed adverse effects of cyclosporin A (CsA) is the development of gingival overgrowth (GO). Fibroblasts are involved in GO, but the question why only a percentage of patients undergoing CsA treatment shows this side-effect remains unanswered. In a previous study, CsA has been demonstrated to induce over-expression of phospholipase C (PLC) beta(1) in fibroblasts of patients with clinical GO, in cells from both enlarged and clinically healthy gingival sites. In this work, we assessed the expression of PLCbeta isoforms to investigate whether the exaggerated fibroblast response to CsA related to increased PLCbeta(1) expression could also be detected in CsA-treated patients without clinical signs of GO. Our results support the hypothesis of a multi-factorial origin of gingival overgrowth, including specific changes within the gingival tissues orchestrating fibroblastic hyper-responsiveness as a consequence of a long-term in vivo exposure to cyclosporin A.

Protein kinase C isoforms undergo quantitative variations during rat spermatogenesis and are selectively retained at specific spermatozoon sites.

Signal transduction elements, including protein kinase C, have been identified in mammalian spermatozoa. In order to evaluate the pattern of expression and the subcellular localization of nine different protein kinase C isoforms in the course of spermatogenesis, we utilized quantitative electron microscopy immunocytochemistry on thin sections of rat seminiferous tubules. The results indicate a progressive reduction of the protein kinase C isoforms present in the early stages of spermatogenesis, so that in late spermatids none of them is present in the nucleus, while the isoforms alpha, gamma and beta II are specifically retained in the acrosome, the isoforms beta I and zeta in the neck, and the isoform epsilon in the tail. These isoforms, except for beta II, are maintained at the same sites in spermatozoa. Western blotting analysis indicates the presence of alpha and gamma isoforms in the head subfraction, and of beta I, zeta and epsilon isoforms in the tail subfraction of spermatozoa. These findings suggest that specific protein kinase C isoforms may be functionally involved in some events of spermatozoa differentiation and, eventually, in the fertilization process.

Nuclear phosphoinositide-specific phospholipase C, phosphatidylinositol 4,5-bisphosphate and protein kinase C during rat spermatogenesis.

Presence and intracellular distribution of phosphoinositide-specific phospholipase C, phosphatidylinositol 4,5-bisphosphate and protein kinase C have been investigated in rat maturing germ cells and spermatozoa. The isoforms beta 1 and gamma 1 of phosphoinositide-specific phospholipase C were immunologically identified and found to be predominantly nuclear or cytoplasmic and nuclear, respectively. The two enzymes were present in the maturing cell lineage of the seminiferous tubule, except for the nucleus of late spermatids, and absent in spermatozoa, in which, however, a phosphoinositide-specific phospholipase C activity persisted, due to yet uncharacterized enzyme(s). Protein kinase C paralleled these developmental changes, and was completely down-regulated in both total cell homogenates and isolated nuclei obtained from spermatozoa. On the contrary, phosphatidylinositol 4,5-bisphosphate, present at the nuclear level in all cell types, accumulated in the nuclei of late spermatids and spermatozoa. These data support the contention that the spermatozoon nucleus stores a lipid-dependent signaling apparatus which could be reactivated either during sperm maturation or at fertilization.

Characterization and cell cycle kinetics of hepatocytes during rat liver regeneration: in vivo BrdUrd incorporation analysed by flow cytometry and electron microscopy.

The incorporation of bromodeoxyuridine (BrdUrd) into newly synthesized DNA has been analysed during hepatocellular regeneration induced by partial hepatectomy in young rats. The kinetic state of the liver has been studied by flow cytometric analysis of the incorporated BrdUrd, while the fine localization of DNA replication sites through the cell cycle has been investigated at the ultrastructural level by the immunogold technique. Eighteen hours after partial hepatectomy flow cytometry revealed an early S phase distribution which corresponded to a specific staining of the interchromatin domains of the hepatocyte nucleus. Thirty-four hours after hepatectomy, on the other hand, when most cells were in late S, a specific staining of heterochromatin domains was observed. The effect of the BrdUrd technique on nuclear aggregation has also been analysed and discussed. The results demonstrate that specific patterns of DNA replication can be recognized during the cell cycle and that flow cytometry and electron microscopy appear to be complementary in the kinetic study of liver regeneration.

Dietary treatment for familial hypercholesterolemia--differential effects of dietary soy protein according to the apolipoprotein E phenotypes.

Familial hypercholesterolemia, one form of type IIa hyperlipidemia, usually responds poorly to standard low-lipid diets. To define the responsiveness to a soy-protein diet in this disease, one homozygous and twenty heterozygous type IIa patients were submitted to a 4-wk traditional hypocholesterolemic diet followed by 4 wk in which animal protein was substituted with texturized soy protein. Soy was then withdrawn for a further 4 wk. No significant changes in plasma lipids were observed during low-lipid diets. The soy diet, however, caused a marked decrease in total (-20.8%) and low-density-lipoprotein (-25.8%) cholesterol and in apolipoprotein B (-14.1%). The plasma cholesterol reduction was higher in patients with apolipoprotein E3/E3 or E3/E4 vs an almost negligible effect on E3/E2. These results confirm that soy-protein diets can lower cholesterol in type IIa patients with familial disease. Data on the sensitivity of patients with different apo-E isoforms agree with recent hypotheses suggesting that soy proteins may activate B,E receptors.

Ultrastructural aspects of the DNA polymerase alpha distribution during the cell cycle.

We studied the nuclear topography of the replicating enzyme DNA polymerase alpha in HeLa cells by transmission electron microscopy and field emission in lens scanning electron microscopy. Cells were synchronized at the G1/S-phase boundary and samples of the different phases of the cell cycle were labeled with an anti-DNA polymerase alpha antibody detected by an immunogold reaction. DNA synthesis was detected by immunogold labeling after bromodeoxyuridine administration. The typical labeling pattern of DNA polymerase alpha observed in G1- and S-phase cells was represented by circular structures 80-100 nm in diameter surrounding an electron-dense area. In double labeled samples these circular structures were associated with bromodeoxyuridine-containing DNA replication sites, forming rosette-like structures. Field emission scanning electron microscopy performed on ultrathin cryosections revealed the chromatin fibers underlying DNA polymerase alpha complexes and showed that the size of the rosette-like structures corresponded to the diameter of chromatin foldings. G2- and M-phase cells showed a spread distribution of DNA polymerase alpha. The evidence of DNA polymerase alpha circular arrangement exclusively in G1- and S-phase cells, obtained by such different approaches, allowed us to consider the three-dimensional structures as DNA replication areas.

Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate localization sites within the nucleus.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key element of signal transduction, being the preferential substrate of specific phospholipases that produce second messengers such as inositol trisphosphate (IP3) and diacylglycerol (DG). Because PIP2 has been cytochemically identified by monoclonal antibodies not only in the cytoplasmic membranes but also in the nuclear envelope and within the nucleus, we performed a study by immunoblotting and by confocal and electron microscopic immunocytochemistry to identify the nuclear sites of PIP2 localization and to exclude any cross-reactivity of the antibody with other nuclear molecules. The results confirm the specificity of the immunolabeling and indicate that PIP2 is localized at precise intranuclear sites both in in situ and in isolated nuclei. They also show that a significant amount of the phospholipid is retained by the cytoskeleton and by the inner nuclear matrix in in situ matrix preparations. Moreover the sensitivity of the immunocytochemical reaction is capable of detecting quantitative variations of PIP2 nuclear content induced by agonists that modulate the signal transduction system at the nuclear level.

Nuclear phospholipase C: a novel aspect of phosphoinositide signalling.

The role of polyphosphoinositides in cellular signalling is well known and recently it has also been shown that the nucleus is a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. It has been demonstrated that phospholipase C specific for inositol lipids (PLC) is one of the main steps of the inositol lipid cycle. The PLC beta family, and especially type beta 1, has given rise to considerable interest since, due to their common COOH-terminus they show nuclear localisation in addition to that at the plasma membrane. It is well established that an autonomous intranuclear inositide cycle exists, and that this cycle is endowed with conventional lipid kinases, phosphatases and PLCs. Among this latter the beta 1 type undergoes stimulation or inhibition under different stimuli and this implicates the beta 1 isoform as a key enzyme for mitogen-activated cell growth as well as for differentiation. Indeed, both the overexpression and the down-regulation of PLC beta 1, by means of antisense mRNA, have demonstrated that PLC plays a role in the nuclear compartment.

Perfluorohexyloctane (F6H8) induces structural modifications and increases apoptosis in rat primary retinal cultures.

The effects of perfluorohexyloctane (F6H8), recently investigated as a long-term artificial vitreous substitute, were studied in vitro, with the use of rat retinal cultures seeded on microporous inserts that allow the cell layer to be in contact with the material to be tested, on the apical side, and with the nutrient medium, on the basal side. After 72 h of treatment with F6H8, retinal cultures lost the characteristic two-layered organization with glial cells at the bottom and neuronal cells on top of them. They appeared to be composed of only one layer of polyhedrical, flattened, and disconnected cells. TUNEL assay revealed an evident increase in the percentage of apoptotic cells in F6H8-treated cultures (30.1 +/- 4.5), compared to control (10.3 +/- 2.6) and perfluoroctane-treated cultures (10.1 +/- 1.7). Immunolabeling of MAP-2, a protein of neuronal cytoskeleton, evidenced a marked loss of neurites. The results suggest that F6H8 is harmful to retinal cells in vitro and can therefore be potentially noxious to the retina as an artificial vitreous substitute.

Curcumin treatment protects rat retinal neurons against excitotoxicity: effect on N-methyl-D: -aspartate-induced intracellular Ca(2+) increase.

Curcumin, an extract from the plant Curcuma longa with well-known antioxidant and anti-inflammatory activities, was tested as protective agent against excitotoxicity in rat retinal cultures. A 24 h-treatment with curcumin reduced N-methyl-D: -aspartate (NMDA)-mediated excitotoxic cell damage, estimated as decrease of cell viability and increase in apoptosis. The protection was associated with decrease of NMDA receptor-mediated Ca(2+) rise and reduction in the level of phosphorylated NR1 subunit of the NMDA receptor. These results enlighten a new pharmacological action of the plant extract, possibly mediated by a modulation of NMDA receptor activity.

In vitro phosphorylation of lamin B by protein kinase C in friend erythroleukemia. Effect of chemically induced differentiation.

Nuclear matrix isolated from murine erythroleukemia cells (Friend cells) has been phosphorylated with gamma 32P-ATP and purified protein kinase C in order to identify specific nuclear substrates for the enzyme. HMBA has been employed to induce the cell to differentiate and to compare the changes of phosphorylation profile after erythroid differentiation. Lamin B has been found to be hyperphosphorylated by rat brain PK-C in nuclear matrix purified from uninduced cells. This difference characterizes the cells from 14 to 72 hrs of HMBA treatment and indicates that the ability of lamin B to be phosphorylated by PK-C is linked to the differentiated state. The involvement of PK-C in lamin phosphorylation might represent an early step of the signalling pathway utilized by erythroid differentiating agents to target the cell nucleus.

Influence of phosphatidylserine on endogeneous RNA synthesis in isolated rat liver nuclei.

The possible involvement of anionic phospholipids in the transcriptional process was studied in isolated rat liver nuclei synthesizing RNA in the presence of phosphatidylserine, which was employed in the form of multillamellar liposomes as a means of delivering the lipid to the nuclei in aqueous medium. The divalent ion requirement for RNA synthesis and the properties of the incubation mixture were not significantly modified by the phospholipid, which increased the rate and the extent of the incorporation of 3H-UMP without changing the endogeneous degradation pattern of the product or affecting the activity of a particular RNA polymerase, as indicated by the sensitivity to amanitin. The thin layer chromatography analysis of the alkaline hydrolysates of the RNA showed that the stimulation involved an increase of the total polyribonucleotide elongation rate. The size of the product was essentially unchanged in the presence of phosphatidylserine, as demonstrated by the qualitative overlapping of the sedimentation profiles of control and lipid treated samples in formamide-sucrose gradients. The release of the H1 fraction from intact nuclei occurring with phosphatidylserine indicated that the DNA template availability was increased by a partial removal of the restrictions imposed by histones, as suggested also by the comparison with heparin and Sarkosyl. These evidences, together with the data accumulated on the occurrence of lipids in chromatin and nuclear matrix, and on their changes related to cell growth, differentiation and malignant transformation, allow a better definition of the role that phospholipids might play in regulating the DNA template availability in the cell.

Electron microscopy microsampling of isolated nuclei sorted by flow cytometry.

This paper describes an efficient method to concentrate for electron microscopic examination minute quantities of subcellular particles obtained by cytofluorimetric sorting. The advantages of this micromethod, based on diafiltration on Millipore filters under constant positive nitrogen pressure, are discussed.

Response of isolated nuclei to phospholipid vesicles: analysis of the nuclear proteins after treatment with phosphatidylserine and phosphatidylcholine and comparison with heparin.

Phospholipid liposomes affect the histone pattern of isolated rat liver nuclei. Multilamellar vesicles (MLV) obtained with phosphatidylserine (PS) release a large amount of the lysine rich histones, while those obtained with phosphatidylcholine (PC) do not induce significant changes with respect to controls. This different response has been compared to the effects obtained with Heparin, which slightly modifies the relative ratio of the histone fractions. These data might account for the mode by which phospholipids induce transitions of the chromatin structure and changes of the endogenous RNA polymerase activity.