Phosphatidylcholine-specific phospholipase C inhibition reduces HER2-overexpression, cell proliferation and tumor growth in a highly tumorigenic ovarian cancer model.

Antagonizing the oncogenic effects of human epidermal growth factor receptor 2 (HER2) with current anti-HER2 agents has not yet yielded major progress in the treatment of advanced HER2-positive epithelial ovarian cancer (EOC). Using preclinical models to explore alternative molecular mechanisms affecting HER2 overexpression and oncogenicity may lead to new strategies for EOC patient treatment. We previously reported that phosphatidylcholine-specific phospholipase C (PC-PLC) exerts a pivotal role in regulating HER2 overexpression in breast cancer cells.

Activation of Phosphatidylcholine-Specific Phospholipase C in Breast and Ovarian Cancer: Impact on MRS-Detected Choline Metabolic Profile and Perspectives for Targeted Therapy.

Elucidation of molecular mechanisms underlying the aberrant phosphatidylcholine cycle in cancer cells plays in favor of the use of metabolic imaging in oncology and opens the way for designing new targeted therapies. The anomalous choline metabolic profile detected in cancer by magnetic resonance spectroscopy and spectroscopic imaging provides molecular signatures of tumor progression and response to therapy. The increased level of intracellular phosphocholine (PCho) typically detected in cancer cells is mainly attributed to upregulation of choline kinase, responsible for choline phosphorylation in the biosynthetic Kennedy pathway, but can also be partly produced by activation of phosphatidylcholine-specific phospholipase C (PC-PLC).

Nuclear phosphoinositide-specific phospholipase C β1 controls cytoplasmic CCL2 mRNA levels in HIV-1 gp120-stimulated primary human macrophages.

HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of CCL2 from macrophages. In turn, this chemokine acts as an autocrine factor enhancing viral replication. In this study, we show for the first time that phosphoinositide-specific phospholipase C (PI-PLC) is required for the production of CCL2 triggered by gp120 in macrophages.

Immune surveillance properties of human NK cell-derived exosomes.

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity.

Inhibition of phosphatidylcholine-specific phospholipase C downregulates HER2 overexpression on plasma membrane of breast cancer cells.

Introduction: Overexpression on plasma membrane of human epidermal growth factor receptor 2 (HER2) is reported in 25% to 30% of breast cancers. Heterodimer formation with cognate members of the epidermal growth factor receptor (EGFR) family, such as HER3 and EGFR, activates abnormal cell-signalling cascades responsible for tumorigenesis and further transcriptional HER2 gene upregulation. Targeting the molecular mechanisms controlling HER2 overexpression and recycling may effectively deactivate this feedback-amplification loop.

LOX-1 as a natural IFN-alpha-mediated signal for apoptotic cell uptake and antigen presentation in dendritic cells.

The identification of molecules responsible for apoptotic cell (AC) uptake by dendritic cells (DCs) and induction of T-cell immunity against AC-associated antigens is a challenge in immunology. DCs differentiated in the presence of interferon-alpha (IFN-alpha-conditioned DCs) exhibit a marked phagocytic activity and a special attitude in inducing CD8(+) T-cell response. In this study, we found marked overexpression of the scavenger receptor oxidized low-density lipoprotein receptor 1 (LOX-1) in IFN-alpha-conditioned DCs, which was associated with increased levels of genes belonging to immune response families and high competence in inducing T-cell immunity against antigens derived from allogeneic apoptotic lymphocytes.

Phosphatidylcholine-specific phospholipase C activation in epithelial ovarian cancer cells.

Elucidation of the mechanisms responsible for aberrant phosphatidylcholine (PC) metabolism in cancer cells may allow identification of novel biomarkers of tumor progression and design of new targeted anticancer therapies. We recently reported up-regulation of PC-specific phospholipases in epithelial ovarian cancer cells (EOC) compared with nontumoral (normal or immortalized) counterparts (EONT). In the present study, we focused, in the same cell systems, on levels, subcellular localization, and activity of PC-specific phospholipase C (PC-PLC), for which a key role in cell proliferation, differentiation, and apoptosis has been shown in several mammalian cells.

Phosphatidylcholine-specific phospholipase C activation is required for CCR5-dependent, NF-kB-driven CCL2 secretion elicited in response to HIV-1 gp120 in human primary macrophages.

CCL2 (MCP-1) has been shown to enhance HIV-1 replication. The expression of this chemokine by macrophages is up-modulated as a consequence of viral infection or gp120 exposure. In this study, we show for the first time that the phosphatidylcholine-specific phospholipase C (PC-PLC) is required for the production of CCL2 triggered by gp120 in human monocyte-derived macrophages (MDMs).

Functional role of phosphatidylcholine-specific phospholipase C in regulating CD16 membrane expression in natural killer cells.

CD16, the low-affinity FcIgG receptor (FcgammaRIIIA), is predominantly expressed in human NK cells. Our recent findings indicate that CD16 expression on the outer membrane surface of NK cells is correlated with the membrane expression of phosphatidylcholine-specific phospholipase C (PC-PLC). In the present study we analyzed the trafficking of CD16 from the plasma membrane to cytoplasmic regions, after stimulation with specific mAb.

Association of dystrobrevin and regulatory subunit of protein kinase A: a new role for dystrobrevin as a scaffold for signaling proteins.

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait.

beta-dystrobrevin, a kinesin-binding receptor, interacts with the extracellular matrix components pancortins.

The dystrobrevins (alpha and beta) are components of the dystrophin-associated protein complex (DPC), which links the cytoskeleton to the extracellular matrix and serves as a scaffold for signaling proteins. The precise functions of the beta-dystrobrevin isoform, which is expressed in nonmuscle tissues, have not yet been determined. To gain further insights into the role of beta-dystrobrevin in brain, we performed a yeast two-hybrid screen and identified pancortin-2 as a novel beta-dystrobrevin-binding partner.

Cyclophosphamide enhances the antitumor efficacy of adoptively transferred immune cells through the induction of cytokine expression, B-cell and T-cell homeostatic proliferation, and specific tumor infiltration.

Purpose: Immunotherapy is a promising antitumor strategy, which can be successfully combined with current anticancer treatments, as suggested by recent studies showing the paradoxical chemotherapy-induced enhancement of the immune response. The purpose of the present work is to dissect the biological events induced by chemotherapy that cooperate with immunotherapy in the success of the combined treatment against cancer. In particular, we focused on the following: (a) cyclophosphamide-induced modulation of several cytokines, (b) homeostatic proliferation of adoptively transferred lymphocytes, and (c) homing of transferred lymphocytes to secondary lymphoid organs and tumor mass.

Expression and role of phosphatidylcholine-specific phospholipase C in human NK and T lymphocyte subsets.

We recently reported evidence of phosphatidylcholine-specific phospholipase C (PC-PLC) involvement in NK cell-mediated cytotoxicity and in lytic granule exocytosis. In the present study, different subpopulations of human PBL were investigated in relation to PC-PLC enzyme expression. While a substantial intracellular amount of PC-PLC was detected in all lymphoid subsets, expression of this enzyme on the outer membrane surface reached high levels only in NK cells, was present at low levels in B lymphocytes and in some TCR gamma/delta T cells and was practically absent in CD4(+) and CD8(+ )T lymphocytes.

ICSBP/IRF-8 differentially regulates antigen uptake during dendritic-cell development and affects antigen presentation to CD4+ T cells.

Interferon consensus sequence-binding protein (ICSBP)/interferon regulatory factor 8 (IRF-8) is a transcription factor that plays critical roles in the differentiation of defined dendritic-cell (DC) populations and in the immune response to many pathogens. In this study, we show that splenic DCs (s-DCs) from ICSBP(-/-) mice are markedly defective in their ability to capture and to present exogenous antigens (Ags) to naive CD4(+) T lymphocytes. We found that CD8alpha(+) DCs and, to a lesser extent, CD8alpha(-) DCs from ICSBP(-/-) mice are impaired at internalizing Ags, either through a receptor-mediated pathway or by macropinocytosis, in spite of having a more immature phenotype than their wild-type (WT) counterparts.

Human dendritic cells following Aspergillus fumigatus infection express the CCR7 receptor and a differential pattern of interleukin-12 (IL-12), IL-23, and IL-27 cytokines, which lead to a Th1 response.

Aspergillus fumigatus is the most prevalent airborne fungal pathogen and causes fatal invasive aspergillosis in immunocompromised patients. Given the essential role of dendritic cells (DC) in initiating and regulating immune responses, we investigated the impact of A. fumigatus conidial infection on human DC.

Expression of alternative transcripts of ferroportin-1 during human erythroid differentiation.

Background And Objectives: Ferroportin-1 (FPN1) is expressed in various types of cells that play critical roles in mammalian iron metabolism and appears to act as an iron exporter in these tissues. The aim of this study was to investigate whether erythroid cells possess specific mechanisms for iron export.

Design And Methods: The expression of FPN1 during human erythroid differentiation, the characterization of alternative transcripts, the modulation by iron and the subcellular localization of this protein were studied.

Alterations of choline phospholipid metabolism in ovarian tumor progression.

Recent characterization of abnormal phosphatidylcholine metabolism in tumor cells by nuclear magnetic resonance (NMR) has identified novel fingerprints of tumor progression that are potentially useful as clinical diagnostic indicators. In the present study, we analyzed the concentrations of phosphatidylcholine metabolites, activities of phosphocholine-producing enzymes, and uptake of [methyl-14C]choline in human epithelial ovarian carcinoma cell lines (EOC) compared with normal or immortalized ovary epithelial cells (EONT). Quantification of phosphatidylcholine metabolites contributing to the 1H NMR total choline resonance (3.

IL-2 induces expression and secretion of IFN-gamma in murine peritoneal macrophages.

We investigated the effect of interleukin (IL)-2, a T cell growth factor capable of activating certain macrophage functions, on interferon (IFN)-gamma expression in resting mouse peritoneal macrophages (PM). IL-2 addition to PM from different mouse strains up-modulated IFN-gamma mRNA and protein secretion. It is notable that endogenous type I and II IFNs did not play any role in the IL-2-mediated effect, as comparable levels of secreted IFN-gamma were observed upon IL-2 stimulation of PM from deficient mice.

Phosphatidylcholine-specific phospholipase C in mitogen-stimulated fibroblasts.

To investigate expression, subcellular localization and mechanisms of translocation of phosphatidylcholine-specific phospholipase C (PC-PLC) during the cell proliferative response, biochemical, immunoblotting, and immunofluorescence analyses were performed on quiescent and mitogen-stimulated NIH-3T3 fibroblasts. Platelet-derived growth factor (PDGF), insulin and 12-O-tetradecanoylphorbol-13-acetate induced, in 10-60 min, PC-PLC translocation from a perinuclear cytoplasmic area to the plasma membrane. Following cell exposure to PDGF (60 min), the overall PC-PLC expression increased up to 2-3x, while the enzyme activity increased 5x in total cell lysates, 2x in the plasma membrane, and 4x in the nucleus; moreover, confocal laser scanning microscopy showed a progressive externalization of PC-PLC on the outer plasma membrane surface and its accumulation in the nuclear matrix.

Dendritic cells derived from BCG-infected precursors induce Th2-like immune response.

Human monocytes can differentiate into dendritic cells (DCs) according to the nature of environmental signals. We tested here whether the infection with the live tuberculosis vaccine bacillus Calmette-Guerin (BCG), which is known to be limited in preventing pulmonary tuberculosis, modulates monocyte and DC differentiation. We found that monocytes infected with BCG differentiate into CD1a- DCs (BCG-DCs) in the presence of granulocyte macrophage-colony stimulating factor and interleukin (IL)-4 and acquired a mature phenotype in the absence of maturation stimuli.

Glucosylceramidase mass and subcellular localization are modulated by cholesterol in Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is characterized by the accumulation of cholesterol and sphingolipids in the late endosomal/lysosomal compartment. The mechanism by which the concentration of sphingolipids such as glucosylceramide is increased in this disease is poorly understood. We have found that, in NPC fibroblasts, the cholesterol storage affects the stability of glucosylceramidase (GCase), decreasing its mass and activity; a reduction of cholesterol raises the level of GCase to nearly normal values.

Effect of human natural killer and gammadelta T cells on the growth of human autologous melanoma xenografts in SCID mice.

Natural killer (NK) cells were first identified for their ability to kill tumor cells of different origin in vitro. Similarly, gammadelta T lymphocytes display strong cytotoxic activity against various tumor cell lines. However, the ability of both the NK and gammadelta cells to mediate natural immune response against human malignant tumors in vivo is still poorly defined.

HIV-1 Nef triggers Vav-mediated signaling pathway leading to functional and morphological differentiation of dendritic cells.

The accessory HIV-1 Nef protein plays a key role in AIDS pathogenesis. We recently demonstrated that exogenous Nef triggers phenotypic and functional differentiation of immature dendritic cells (DCs). Here we investigated whether the Nef-induced DC differentiation occurs with morphological remodeling and have focused on the interference of Nef in the signaling pathways that regulates DC maturation.

Triacsin C inhibits the formation of 1H NMR-visible mobile lipids and lipid bodies in HuT 78 apoptotic cells.

Nuclear magnetic resonance-visible mobile lipids (ML) have been reported to accumulate during cell apoptosis in vitro and in vivo. The biogenesis, biochemical nature and structure of these lipids are still under debate. In this study, a human lymphoblastoid cell line, HuT 78, was induced to apoptosis by exposure to anti-Fas monoclonal antibodies (alpha-Fas mAb) followed by incubation for different time intervals (1-24 h, hypodiploid cell fraction, H, varying from 1% to over 60%) either in the presence or in the absence of 5.