Nuclear phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-kinase, Akt, and PTen: emerging key regulators of anti-apoptotic signaling and carcinogenesis.

Inositol lipid-derived second messengers have long been known to have an important regulatory role in cell physiology. Phosphatidylinositol 3-kinase (PI3K) synthesizes the second messenger 3,4,5'-phosphatidylinositol trisphosphate (Ptdlns 3,4,5P3) which controls a multitude of cell functions. Down-stream of PI3K/PtdIns 3,4,5P3 is the serine/threonine protein kinase Akt (protein kinase B, PKB). Since the PI3K/ PtdIns 3,4,5P3 /Akt pathway stimulates cell proliferation and suppresses apoptosis, it has been implicated in carcinogenesis. The lipid phosphatase PTEN is a negative regulator of this signaling network. Until recently, it was thought that this signal transduction cascade would promote its anti-apoptotic effects when activated in the cytoplasm. Several lines of evidence gathered over the past 20 years, have highlighted the existence of an autonomous nuclear inositol lipid cycle, strongly suggesting that lipids are important components of signaling pathways operating at the nuclear level. PI3K, PtdIns(3,4,5)P3, Akt, and PTEN have been identified within the nucleus and recent findings suggest that they are involved in cell survival also by operating in this organelle, through a block of caspase-activated DNase and inhibition of chromatin condensation. Here, we shall summarize the most updated and intriguing findings about nuclear PI3K/ PtdIns(3,4,5)P3/Akt/PTEN in relationship with carcinogenesis and suppression of apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL) and erythropoiesis: a role for PKC epsilon.

The regulation of the hematopoietic stem cell pool size and the processes of cell differentiation along the hematopoietic lineages involve apoptosis. Among the different factors with a recognized activity on blood progenitor cells, TRAIL - a member of the TNF family of cytokines - has an emerging role in the modulation of normal hematopoiesis.PKC(epsilon) levels are regulated by EPO in differentiating erythroid progenitors and control the protection against the apoptogenic effect of TRAIL. EPO-induced erythroid CD34 cells are insensitive to the apoptogenic effect of TRAIL between day 0 and day 3, due to the lack of specific surface receptors expression. Death receptors appear after day 3 of differentiation and consequently erythroid cells become sensitive to TRAIL up to day 9/10, when the EPO-driven up-regulation of PKC epsilon intracellular levels inhibits the TRAIL-mediated apoptosis, via Bcl-2. In the time interval between day 3 and 9, therefore, the number of erythroid progenitors can be limited by the presence of soluble or membrane-bound TRAIL present in the bone marrow microenvironment.

Nuclear envelope proteins and chromatin arrangement: a pathogenic mechanism for laminopathies.

The involvement of the nuclear envelope in the modulation of chromatin organization is strongly suggested by the increasing number of human diseases due to mutations of nuclear envelope proteins. A common feature of these diseases, named laminopathies, is the occurrence of major chromatin defects. We previously reported that cells from laminopathic patients show an altered nuclear profile, and loss or detachment of heterochromatin from the nuclear envelope. Recent evidence indicates that processing of the lamin A precursor is altered in laminopathies featuring pre-mature aging and/or lipodystrophy phenotype. In these cases, pre-lamin A is accumulated in the nucleus and heterochromatin is severely disorganized. Here we report evidence indicating that pre-lamin A is mis-localized in the nuclei of Emery-Dreifuss muscular dystrophy fibroblasts, either bearing lamin A/C or emerin mutations. Abnormal pre-lamin A-containing structures are formed following treatment with a farnesyl-transferase inhibitor, a drug that causes accumulation of pre-lamin A. Pre-lamin A-labeled structures co-localize with heterochromatin clumps. These data indicate that in almost all laminopathies the expression of the mutant lamin A precursor disrupts the organization of heterochromatin domains. Our results further show that the absence of emerin expression alters the distribution of pre-lamin A and of heterochromatin areas, suggesting a major involvement of emerin in pre-lamin A-mediated mechanisms of chromatin remodeling.

Chromatin phospholipids in normal and chronic lymphocytic leukemia lymphocytes.

Certain phospholipids are associated with the nonhistone chromosomal proteins extracted from normal B- and chronic lymphocytic leukemia lymphocytes. The ratio of phospholipids to nonhistone chromosomal proteins was constant with the different methods used for isolating nuclei and extracting the chromatin, although the various methods allowed a different recovery of total lipids from chromatin. Three phospholipids were extractable from the nonhistone protein fraction, but their respective ratios varied in chronic lymphocytic leukemia compared to normal B-lymphocytes. The most significant variation concerns the reduction of sphingomyelin content in leukemic lymphocytes, since this prospholipid in vitro affects both DNA stability and transcription.

Uptake and phosphorylation of phosphatidylinositol by rat liver nuclei. Role of phosphatidylinositol transfer protein.

The incorporation of phosphatidyl[2-3H]inositol ([3H]PI) from vesicles or microsomal membranes into rat liver nuclei is greatly stimulated by phosphatidylinositol transfer protein (PI-TP). The nuclei are able to phosphorylate [3H]PI, with the production of phosphatidylinositol 4-phosphate (PIP). Recovery of tritiated inositol trisphosphate, inositol phosphate, glycerophosphoinositol and inositol, suggests that in isolated nuclei a large set of enzymes of the PI cycle is present, similar to the enzymes involved in the plasma membrane PI cycle. Incubation with [gamma-32P]ATP shows that isolated nuclei are able to phosphorylate endogenous PI to PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). In the presence of exogenous PI and detergent the synthesis of PIP is increased, indicating that in nuclei the PI pool is suboptimal for the PI-kinase activity. The present study suggests that PI-TP may be involved in providing substrates for PI metabolism at the nuclear level.

Lipid phosphorylation in isolated rat liver nuclei. Synthesis of polyphosphoinositides at subnuclear level.

Isolated rat liver nuclei and subnuclear fractions synthesize polyphosphoinositides in vitro in a mode dependent on the presence of nuclear membrane, detergent and exogenous substrates. The nuclear membrane is not essential as a source of lipid kinases, since the addition of exogenous phosphatidylinositol or phosphatidylinositol monophosphate to reaction mixtures lacking membranes restores the synthesis of phosphatidylinositol mono- and bisphosphate, respectively. Inositide phosphorylation is best accomplished by high-salt extracted nuclei and pre-detergent lamina. These data suggest that the nucleus, and especially the nuclear periphery, is a cell compartment in which polyphosphoinositide synthesis occurs; this might be related to the progression of phosphatidylinositol metabolism-dependent signals to the genetic apparatus.

Natural killer function in flow cytometry. I. Evaluation of NK lytic activity on K562 cell line.

Flow cytometry has been employed to establish an NK assay using the K562 target cell line. These cells show a perpendicular light scatter (PLS) characteristically different from lymphocytes. Other physical parameters, such as forward light scatter (FLS), do not discriminate between the two populations, since dead K562 cells display similar FLS characteristics as effector cells. Killed cells are stained with propidium iodide and followed by flow analysis. It was advisable to add the dye in the medium so that, as long as the target cells are killed they will also be stained. Moreover the flow cytometric and trypan blue evaluation of target cell death rate showed a stronger correlation than did either test with the conventional 51Cr release assay, the first two methods both being based on the same biological mechanism.

Use of poligonal windows for physical discrimination among mononuclear subpopulations in flow cytometry.

Peripheral blood mononuclear cells from ten healthy normal donors were analyzed by means of monoclonal antibodies and flow cytometry (FACS IV). In order to apply optimal gating for the identification and exclusion of monocytes from lymphocyte populations, mononuclear cells were analyzed on the scattergram using rectangular or poligonal computer-generated windows. Two main windows on lymphoid population were used which mainly differed for the up-right corner, in the border area between lymphocytes and monocytes. While the number of lymphocytes contained either in the regular or in the poligonal windows was the same, the number of contaminating monocytes decreased by two-fold in the poligonal one. Besides, the use of tight lymphocyte gating, in order to reach lower monocyte contamination, leads to a loss of lymphoid cells which does not appear to be random, but seems to affect mainly the Leu11c+ population with natural killer activity. These cells produce forward and perpendicular scatter signals higher than other lymphocyte subsets, and, therefore, are mainly located in the area of the scattergram which divides lymphocytes from monocytes. These data are in accordance with the large granular morphology of natural killer cells. The use of the poligonal windows seems to be useful to reduce monocyte contamination with no selective loss of natural killer lymphocytes, and may be particularly helpful in the analysis of pathological samples.

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.

Intranuclear localization of phospholipids by ultrastructural cytochemistry.

The presence of phospholipids within the interphase nucleus and in isolated chromatin, previously demonstrated by analytical biochemical methods, has been only rarely documented by cytochemical procedures, especially at the ultrastructural level. By means of a gold-conjugated phospholipase technique, we investigated the fine localization of endogenous phospholipids in the different nuclear domains in rat pancreas and in cell cultures. To reduce possible removal or displacement of phospholipids, different specimen preparation procedures such as cryofixation, cryosectioning, and freeze-fracturing were utilized. Apart from slight differences in efficiency among these methods, phospholipids have been cytochemically identified in the same nuclear domains: the interchromatin granules and fibers and the dense fibrillar component of the nucleolus. These results suggest that the phospholipids are an actual nuclear component, not randomly distributed in the nucleoplasm but mainly localized in the nuclear domains involved in the synthesis, maturation, and transport of ribonucleoproteins.

Chromatin structural transitions following histone H1 displacement by phosphatidylserine vesicles and low pH treatment. A multiparametric analysis involving flow cytometry, electron microscopy, and nuclease digestion.

We describe several morphological and functional modifications in isolated rat liver nuclei incubated in the presence of phosphatidylserine (PS) multilamellar vesicles (MLV). These effects, which occur through the release of histone H1, induce chromatin decondensation, as shown by electron microscopy and nuclease digestion. Flow cytometry was employed to monitor these changes in chromatin structure in isolated nuclei by means of perpendicular light scatter (PLS) and fluorescence signals. Chromatin decondensation induced by PS or by low pH treatment was accompanied by an increase in perpendicular light scatter and by less efficient binding of ethidium bromide. These flow cytometric findings are peculiar to chromatin decondensation induced by displacement of histone H1. Conversely, chromatin decondensation caused by lowering of the divalent ion concentration, without displacement of histone H1, is characterized only by an increase in perpendicular light scatter.

Distribution of acid alpha-naphthyl acetate esterase among human T lymphocyte subsets.

Human T lymphocyte subsets, identified by means of OKT3, 4 and 8 monoclonal antibodies, were isolated by a fluorescence activated cell sorter (FACS IV) and analyzed for distribution of alpha-naphthyl acetate esterase (ANAE) activity. As compared to OKT8+ lymphocytes a higher proportion of OKT4+ lymphocytes was ANAE-positive exhibiting a spot or dot-like pattern in the cytoplasm. OKT8 and 4 positive subsets showed a similar ANAE distribution in diffuse granular form. Although OKT4 and OKT8 populations presented a different ANAE dot-like reactivity, this marker did not allow as clear a distinction between them as that reported for TG and TM lymphocytes.

Impaired lymphocyte stimulation induced by long-term training.

In recent there has been increasing interest in the definition of hormone influence on the immune system. Physical stress provides a suitable model for studying the interactions between the immune system and the neuroendocrine factors which have been shown to modulate the lymphoid cellular compartment. Our approach has been devoted to defining the stable modifications induced in the immune system in athletes during agonistic training. The results show that the circulating compartment of the immune system tends to modulate its different subsets under the continuous influence of stress hormones, together with a specific functional impairment of the helper subset in the proliferative response after stimulation with PHA, and particularly with PWM.

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.

Inositol lipid phosphorylation in the cell nucleus.

Inositol lipid metabolism has been analyzed in isolated rat liver nuclei and nuclear fractions, in order to determine the subcellular distribution of the sites of lipid phosphorylation and breakdown. Lipid kinases and phosphoesterases appear to be tightly bound nuclear components, and can utilize exogenous substrates administered to membrane-depleted structures. The possible involvement of specific carrier protein in the nuclear metabolism of inositol lipids has also been analysed by studying the uptake and processing of phosphatidylinositol transferred to the isolated nuclei by phosphatidylinositol transfer protein (PI-TP). PI-TP greatly stimulates the incorporation of phosphatidylinositol from microsomal membranes and synthetic vesicles, and the lipid taken up is available for phosphorylation and breakdown by enzymes associated to the nucleus. The results obtained support previous data on the metabolic and structural role of nuclear lipids, and suggest that the cell nucleus is a site of lipid phosphorylation, not necessarily involving enzymes and substrates located on the nuclear membrane. They also indicate that an integrated signalling pathway can exist at the nuclear level utilizing inositol lipid-derived second messengers and PKC to control replication and transcription.

Unfolding of nucleosome core induced by phosphatidylserine.

The main experimental findings on the actual presence of lipids among the minor chromatin components are revised and discussed especially in the light of the reported effects that exogenous lipids induce in DNA and RNA synthesis by using purified templates. Moreover, all the available evidence of the influence of phospholipid liposomes on the activities and structure of isolated nuclei are reported. In order to further clarify the possible mechanism by which phospholipids could affect gene expression, the modifications at the nucleosome core level have been investigated by means of IAF staining and electron microscopy. The results obtained indicate that the increased transcriptional activity induced by PS MLV in isolated nuclei requires both the removal of histone H1, which causes the unfolding of the solenoid into the nucleosome fiber configuration of the chromatin, and the subsequent splitting of the H3 dimer. This latter process, monitored by IAF accessibility to H3 in isolated nucleosomes incubated with PS, causes the transition from the nucleosome to the lexosome structure, which is the configuration favoring the activity of RNA polymerases.

Effect of phospholipids on transcription and ribonucleoprotein processing in isolated nuclei.

The response of isolated rat liver and murine erythroleukemia nuclei to phospholipid liposomes has been monitored with different techniques, by studying the endogenous RNA synthesis, the release of transcripts in the medium, the pattern of acid-extractable nuclear proteins and the ultra-structural morphology. Total transcription in rat liver and beta-globin mRNA synthesis in MEL nuclei are increased by PS and reduced by PC. These changes of RNA polymerase activity, and the transport of RNAs from nucleus as well as the nuclear protein changes, correlate with structural transitions which occur in both types of nuclei, consisting of euchromatization with loss of RNP particles in the case of PS and opposite effects with PC. The significance of these modifications in relationship to the possible involvement of phospholipids in the control of gene expression is discussed.

Lipid mediated signal transduction in the cell nucleus.

Cell growth and differentiation can be affected by the transduction of extracellular signals involving cyclic nucleotides, inositol phospholipids and phospholipid dependent protein kinase C systems. Since we previously reported existence of lipids inside the nucleus and nuclear fractions, it seems of interest to examine the possible presence of the cascade of inositol lipids in isolated nuclei as well as the presence of the protein kinase C, whose activity is tightly related to the phosphoinositide cycle, and requires the presence of phosphatidylserine, which has been previously demonstrated to deeply affect nuclear structure and function. Here we show that highly purified nuclei from both rat liver and Friend cells, free of nuclear membrane, can incorporate radiolabel from ATP-[32P] into phosphatidic acid, phosphatidyl-inositol phosphate and phosphatidylinositol (4', 5')bisphosphate. The degree of radiolabelling of phosphatidylinositol bisphosphate is highly dependent on the state of differentiation of the cells. Moreover, a doublet of immunoreactive bands has been identified in rat liver nuclei by means of a polyclonal antibody against protein kinase C. The two polypeptides appear to be tightly bound to the nuclear matrix. These two forms of the enzyme might be translational products specifically located in the nucleus, involved in the transduction to the genomic apparatus of regulatory signals generated by growth factors and tumor promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Changes in inositol lipid metabolism and protein kinase C translocation in nuclei of mitogen stimulated Swiss 3T3 cells.

The correlation between changes in nuclear polyphosphoinositide levels preceding PKC translocation to the nucleus and the onset of DNA synthesis has been discussed. Using two different clones of Swiss 3T3 fibroblasts belonging to the same original cell line, one of which is unresponsive to mitogenic stimulation with IGF-I on its own or in combination with bombesin, it has been observed that a rapid and transient breakdown of nuclear PIP and PIP2 occurs only in responsive cells and this precedes the translocation of PKC to the nucleus, as evidenced by immunochemical analysis as well as by enzymatic activity. Therefore, it seems that a direct link exists between nuclear polyphosphoinositide metabolism, PKC translocation to the nucleus and cell division. Since IGF-I acts at the plasma membrane through a tyrosine kinase receptor it seems that the mitogenic stimulation induced by this factor utilizes different signalling pathways at the plasma membrane and at the nucleus. Because of the evidence that type I IGF receptor is expressed in both responsive and unresponsive cells and that the receptor machinery at the plasma membrane is active the lack of the transient changes in nuclear inositol lipids and of PKC translocation in unresponsive cells further suggests that the cell nucleus is capable of an autonomous signalling system based on polyphosphoinositide metabolism.