Forebrain-specific ablation of phospholipase Cγ1 causes manic-like behavior.

Manic episodes are one of the major diagnostic symptoms in a spectrum of neuropsychiatric disorders that include schizophrenia, obsessive-compulsive disorder and bipolar disorder (BD). Despite a possible association between BD and the gene encoding phospholipase Cγ1 (PLCG1), its etiological basis remains unclear. Here, we report that mice lacking phospholipase Cγ1 (PLCγ1) in the forebrain (Plcg1f/f; CaMKII) exhibit hyperactivity, decreased anxiety-like behavior, reduced depressive-related behavior, hyperhedonia, hyperphagia, impaired learning and memory and exaggerated startle responses. Inhibitory transmission in hippocampal pyramidal neurons and striatal dopamine receptor D1-expressing neurons of Plcg1-deficient mice was significantly reduced. The decrease in inhibitory transmission is likely due to a reduced number of γ-aminobutyric acid (GABA)-ergic boutons, which may result from impaired localization and/or stabilization of postsynaptic CaMKII (Ca2+/calmodulin-dependent protein kinase II) at inhibitory synapses. Moreover, mutant mice display impaired brain-derived neurotrophic factor-tropomyosin receptor kinase B-dependent synaptic plasticity in the hippocampus, which could account for deficits of spatial memory. Lithium and valproate, the drugs presently used to treat mania associated with BD, rescued the hyperactive phenotypes of Plcg1f/f; CaMKII mice. These findings provide evidence that PLCγ1 is critical for synaptic function and plasticity and that the loss of PLCγ1 from the forebrain results in manic-like behavior.

Global Longitudinal Strain for the Early Detection of Chemotherapy-Induced Cardiotoxicity: A Systematic Review and Meta-analysis.

AIMS: Left ventricular ejection fraction is used to monitor patients undergoing cardiotoxic chemotherapy. A decrease in left ventricular ejection fraction represents a relatively late stage of systolic involvement. Global longitudinal strain has been studied to detect early changes in left ventricular myocardial contractile function. The aim of the present study was to evaluate the global longitudinal strain measurement in the early detection of cardiotoxicity induced by cardiotoxic chemotherapeutic agents. MATERIALS AND METHODS: A study search strategy based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) was carried out to report systematic reviews. A search on PubMed, EMBASE, Web of Science and SCOPUS was carried out using the following keywords: 'echocardiography' and 'cardiotoxicity' and their variations, without language or date restrictions (until March 2021). RESULTS: In total, 4873 articles were identified for title and abstract analysis. The systematic review included 10 studies comprising 661 patients with cancer, including mainly breast cancer and haematological malignancies, mainly treated with anthracyclines. The meta-analysis included four studies: patients with cardiotoxicity showed a reduction in strain, compared with baseline, 14.13% greater than patients without cardiotoxicity (95% confidence interval 5.07-23.19; P < 0.01). No heterogeneity was observed between studies (I2 = 0). CONCLUSION: The meta-analysis showed that strain is a tool with proper predictive capacity for the detection of cardiotoxicity.

Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes activated after stimulation with lipopolysaccharide.

Signal transduction pathways, involved in cell cycle and activities, depend on various components including lipid signalling molecules, such as phosphoinositides and related enzymes. Many evidences support the hypothesis that inositol lipid cycle is involved in astrocytes activation during neurodegeneration. Previous studies investigated the pattern of expression of phosphoinositide-specific phospholipase C (PI-PLC) family isoforms in astrocytes, individuating in cultured neonatal rat astrocytes, supposed to be quiescent cells, the absence of some isoforms, accordingly to their well known tissue specificity. The same study was conducted in cultured rat astrocytoma C6 cells and designed a different pattern of expression of PI-PLCs in the neoplastic counterpart, accordingly to literature suggesting a PI signalling involvement in tumour progression. It is not clear the role of PI-PLC isoforms in inflammation; recent data demonstrate they are involved in cytokines production, with special regard to IL-6. PI-PLCs expression in LPS treated neonatal rat astrocytes performed by using RT-PCR, observed at 3, 6, 18 and 24 h intervals, expressed: PI-PLC beta1, beta4 and gamma1 in all intervals analysed; PI-PLC delta1 at 6, 18 and 24 h; PI-PLC delta3 at 6 h after treatment. PI-PLC beta3, delta4 and epsilon, present in untreated astrocytes, were not detected after LPS treatment. Immunocytochemical analysis, performed to visualize the sub-cellular distribution of the expressed isoforms, demonstrated different patterns of localisation at different times of exposure. These observations suggest that PI-PLCs expression and distribution may play a role in ongoing inflammation process of CNS.

Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts.

A high incidence of relapses following induction chemotherapy is a major hindrance to patient survival in acute myelogenous leukemia (AML). There is strong evidence that activation of the phosphoinositide 3 kinase (PI3K)/Akt signaling network plays a significant role in rendering AML blasts drug resistant. An important mechanism underlying drug resistance is represented by overexpression of membrane drug transporters such as multidrug resistance-associated protein 1 (MRP1) or 170-kDa P-glycoprotein (P-gp). Here, we present evidence that MRP1, but not P-gp, expression is under the control of the PI3K/Akt axis in AML blasts. We observed a highly significant correlation between levels of phosphorylated Akt and MRP1 expression in AML cells. Furthermore, incubation of AML blasts with wortmannin, a PI3K pharmacological inhibitor, resulted in lower levels of phosphorylated Akt, downregulated MRP1 expression, and decreased Rhodamine 123 extrusion in an in vitro functional dye efflux assay. We also demonstrate that wortmannin-dependent PI3K/Akt inhibition upregulated p53 protein levels in most AML cases, and this correlated with diminished MRP1 expression and enhanced phosphorylation of murine double minute 2 (MDM2). Taken together, these data suggest that PI3K/Akt activation may lead to the development of chemoresistance in AML blasts through a mechanism involving a p53-dependent suppression of MRP1 expression.

Targeting the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin module for acute myelogenous leukemia therapy: from bench to bedside.

The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B, PKB)/mammalian Target Of Rapamycin (mTOR) signaling pathway plays a critical role in many cellular functions which are elicited by extracellular stimuli. However, constitutively active PI3K/Akt/mTOR signaling has also been firmly established as a major determinant for cell growth, proliferation, and survival in an wide array of human cancers. Thus, blocking the PI3K/AKT/mTOR signal transduction network could be an effective new strategy for targeted anticancer therapy. Pharmacological inhibitors of this signaling cascade are powerful antineoplastic agents in vitro and in xenografted models of tumors, and some of them are now being tested in clinical trials. Recent studies showed that PI3K/Akt/mTOR axis is frequently activated in acute myelogenous leukemia (AML) patient blasts and strongly contributes to proliferation, survival, and drug-resistance of these cells. Both the disease-free survival and overall survival are significantly shorter in AML cases with PI3K/Akt/mTOR upregulation. Therefore, this signal transduction cascade may represent a target for innovative therapeutic treatments of AML patients. In this review, we discuss the possible mechanisms of activation of this pathway in AML cells and the downstream molecular targets of the PI3K/Akt/mTOR signaling network which are important for blocking apoptosis, enhancing proliferation, and promoting drug-resistance of leukemic cells. We also highlight several pharmacological inhibitors which have been used to block this pathway for targeted therapy of AML. These small molecules induce apoptosis or sensitize AML cells to existing drugs, and might be used in the future for improving the outcome of this hematological disorder.

Phosphorylation of nuclear phospholipase C beta1 by extracellular signal-regulated kinase mediates the mitogenic action of insulin-like growth factor I.

It is well established that a phosphoinositide (PI) cycle which is operationally distinct from the classical plasma membrane PI cycle exists within the nucleus, where it is involved in both cell proliferation and differentiation. However, little is known about the regulation of the nuclear PI cycle. Here, we report that nucleus-localized phospholipase C (PLC) beta1, the key enzyme for the initiation of this cycle, is a physiological target of extracellular signal-regulated kinase (ERK). Stimulation of Swiss 3T3 cells with insulin-like growth factor I (IGF-I) caused rapid nuclear translocation of activated ERK and concurrently induced phosphorylation of nuclear PLC beta1, which was completely blocked by the MEK inhibitor PD 98059. Coimmunoprecipitation detected a specific association between the activated ERK and PLC beta1 within the nucleus. In vitro studies revealed that recombinant PLC beta1 could be efficiently phosphorylated by activated mitogen-activated protein kinase but not by PKA. The ERK phosphorylation site was mapped to serine 982, which lies within a PSSP motif located in the characteristic carboxy-terminal tail of PLC beta1. In cells overexpressing a PLC beta1 mutant in which serine 982 is replaced by glycine (S982G), IGF-I failed to activate the nuclear PI cycle, and its mitogenic effect was also markedly attenuated. Expression of S982G was found to inhibit ERK-mediated phosphorylation of endogenous PLC beta1. This result suggests that ERK-evoked phosphorylation of PLC beta1 at serine 982 plays a critical role in the activation of the nuclear PI cycle and is also crucial to the mitogenic action of IGF-I.

Phosphoinositide 3-kinase/Akt signaling pathway and its therapeutical implications for human acute myeloid leukemia.

The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is crucial to many aspects of cell growth, survival and apoptosis, and its constitutive activation has been implicated in the both the pathogenesis and the progression of a wide variety of neoplasias. Hence, this pathway is an attractive target for the development of novel anticancer strategies. Recent studies showed that PI3K/Akt signaling is frequently activated in acute myeloid leukemia (AML) patient blasts and strongly contributes to proliferation, survival and drug resistance of these cells. Upregulation of the PI3K/Akt network in AML may be due to several reasons, including FLT3, Ras or c-Kit mutations. Small molecules designed to selectively target key components of this signal transduction cascade induce apoptosis and/or markedly increase conventional drug sensitivity of AML blasts in vitro. Thus, inhibitory molecules are currently being developed for clinical use either as single agents or in combination with conventional therapies. However, the PI3K/Akt pathway is important for many physiological cellular functions and, in particular, for insulin signaling, so that its blockade in vivo might cause severe systemic side effects. In this review, we summarize the existing knowledge about PI3K/Akt signaling in AML cells and we examine the rationale for targeting this fundamental signal transduction network by means of selective pharmacological inhibitors.

Frequent elevation of Akt kinase phosphorylation in blood marrow and peripheral blood mononuclear cells from high-risk myelodysplastic syndrome patients.

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myeloid leukemia (AML). The progression of myelodysplastic syndromes (MDSs) to AML is thought to be associated with abrogation of apoptotic control mechanisms. However, little is known about signal transduction pathways which may be involved in enhanced survival of MDS cells. In this report, we have performed immunocytochemical and flow cytometric analysis to evaluate the levels of activated Akt in bone marrow or peripheral blood mononuclear cells from patients diagnosed with MDS. We observed high levels of Ser473 phosphorylated Akt (p-Akt) staining in 90% of the cases (n=22) diagnosed as high-risk MDS, whereas mononuclear cells from normal bone marrow or low-risk MDS patients showed low or absent Ser473 p-Akt staining. Furthermore, all high-risk MDS patients also demonstrated high expression of the Class I PI3K p110delta catalytic subunit and a decreased expression of PTEN. Taken together, our results suggest that Akt activation might be one of the factors contributing to the decreased apoptosis rate observed in patients with high-risk MDS.

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.

Nuclear expression of diacylglycerol kinases: possible involvement in DNA replication.

The existence of intranuclear lipid-dependent signal transduction systems has been demonstrated by several independent groups. Remarkably, intranuclear lipid-dependent signal transduction pathways are regulated independently from their membrane/cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation, differentiation, and apoptosis. Diacylglycerol (DG) is a fundamental lipid second messenger which is produced in the nucleus. Since the levels of nuclear DG fluctuate during the cell cycle progression, it has been suggested that this lipid second messenger has important regulatory roles. Most likely, nuclear DG serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases (DGKs), i.e. the enzymes that, by converting DG into phosphatidic acid (PA), terminate DG-dependent events. This review aims at highlighting the different isozymes of DGKs present within the nucleus as well as at discussing their potential functions with particular emphasis placed on DNA replication.

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.

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.

Essential role for nuclear phospholipase C beta1 in insulin-like growth factor I-induced mitogenesis.

The nucleus has been shown to be a site for the inositol lipid cycle that can be affected by treatment of quiescent cells with growth factors such as insulin-like growth factor I (IGF-I). Indeed, the exposure of Swiss 3T3 cells to IGF-I results in a rapid and transient increase in nuclear phospholipase C (PLC) beta1 activity. In addition, several other reports have shown the involvement of PLC beta1 in nuclear signaling in different cell types. Although the demonstration of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate hydrolysis by nuclear PLC beta1 established the existence of nuclear PLC signaling, the significance of this autonomous pathway in the nucleus has yet to be thoroughly clarified. By inducing both the inhibition of PLC beta1 expression by antisense RNA and its overexpression, we show that this nuclear PLC is essential for the onset of DNA synthesis following IGF-I stimulation of quiescent Swiss 3T3 cells.

Enhanced nuclear diacylglycerol kinase activity in response to a mitogenic stimulation of quiescent Swiss 3T3 cells with insulin-like growth factor I.

Results from several laboratories have established the existence in the nucleus of an autonomous polyphosphoinositide cycle, which is involved in both cell proliferation and differentiation. A key step of intranuclear polyphosphoinositide metabolism is the phospholipase C-mediated generation of diacylglycerol (DAG). In insulin-like growth factor (IGF)-I-stimulated Swiss 3T3 cells, a transient elevation of intranuclear DAG levels is essential for attracting the alpha isoform of protein kinase C (PKC) to the nucleus. Previous evidence has shown that the nucleus also contains DAG kinase, i.e., the enzyme that yields phosphatidic acid from DAG, thus terminating PKC-mediated signaling events. Here we show that IGF-I treatment of quiescent Swiss 3T3 cells results in the stimulation of nuclear DAG kinase activity. Time course analysis showed an inverse relationship between nuclear DAG mass and DAG kinase activity levels. After IGF-I treatment, maximal enhancement of DAG kinase activity was measured in the internal matrix domain of the nucleus. PKC-alpha remained within the nuclear compartment, even when nuclear DAG mass returned to basal levels. This was conceivably due to interactions with specific nuclear PKC-binding proteins, some of which were identified as lamins A, B, and C and protein C23/nucleolin. Treatment of cells with two DAG kinase inhibitors, R59022 and R59949, blocked the IGF-I-dependent rise in nuclear DAG kinase activity and maintained elevated intranuclear levels of DAG. The two inhibitors also markedly potentiated the mitogenic effect of IGF-I. These results suggest that nuclear DAG kinase plays a key role in regulating the levels of DAG present in the nucleus and that DAG is a key molecule for the mitogenic effect that IGF-I exerts on Swiss 3T3 cells.

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.

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.

Inhibition of the EGF-induced activation of phospholipase C-gamma1 by a single chain antibody fragment.

Phospholipase C-gamma1(PLC-gamma1) is known to play an essential role in various cellular responses, such as proliferation and tumorigenesis, and PLC-gamma1-specific inhibitors are commonly employed to investigate the mechanism of the PLC-gamma1-mediated signaling pathway. In this study, we developed a single chain antibody fragment (scFv) as a blocker for PLC-gamma1 mediated signaling. scFv, designated F7-scFv, specifically bound to PLC-gamma1 with high affinity (K(d)=1.9x10(-8) M) in vitro. F7-scFv also bound to PLC-gamma1 in vivo and altered the distribution pattern of PLC-gamma1 from the cytoplasm to the intracellular aggregates, where F7-scFv was localized. Moreover, F7-scFv interrupted the EGF-induced translocation of PLC-gamma1 from the cytosol to the membrane ruffle and attenuated EGF-induced inositol phosphates generation and intracellular calcium mobilization. These results indicate that F7-scFv blocks EGF-induced PLC-gamma1 activation by causing sequestering of PLC-gamma1 into intracellular aggregates, and may therefore be useful in studies of the PLC-gamma1-mediated signaling pathway.

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.