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Biomedical subjects

A Santoni

Publications and source records attributed to A Santoni.

At least 55 records · Page 3Linked to original sources
PubMed1997

Selective binding of shc-SH2 domain to tyrosine-phosphorylated zeta but not gamma-chain upon CD16 ligation on human NK cells.

Fc gamma RIII (CD16) is a hetero-oligomeric receptor composed of a ligand-binding alpha subunit associated with homo- or heterodimers of the TCR zeta- and Fc epsilon RI gamma-chains. We have previously demonstrated that CD16 ligation promotes complex formation between tyrosine-phosphorylated shc and Grb2, leading to activation of ras signaling pathway in human NK cells. Here we report that CD16 engagement induces rapid shc association with the tyrosine-phosphorylated receptor complex in human NK cells. In vitro binding studies demonstrate that this interaction is mediated by the shc-SH2 domain, and immunodepletion experiments indicate that the zeta- but not the gamma-chain has the capability to mediate this association. Jurkat cell clones expressing CD16-zeta or -gamma homodimers have been used to gain more information about the mechanism of shc/CD16 association. Our data show that, while engagement of both receptors induces tyrosine phosphorylation of shc and Grb2 recruitment, shc-SH2/receptor complex association is evident only in CD16-zeta but not in CD16-gamma transfectants. Overall, our data demonstrate that the adaptor protein shc can be recruited to the activated CD16 complex by interaction with tyrosine-phosphorylated zeta-chain in a SH2-dependent manner. These results also provide further support to the notion that zeta- and gamma-chains might couple to different biochemical pathways.

Cells, Cultured↗
PubMed1997

Functional role of alpha4beta1 and alpha5beta1 integrin fibronectin receptors expressed on adriamycin-resistant MCF-7 human mammary carcinoma cells.

Cytofluorimetric and reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that adriamycin-resistant (ADRR), but not sensitive (WT), MCF-7 human mammary carcinoma cell lines express alpha4beta1 and alpha5beta1 integrins. ADR(R) cells adhere to fibronectin (FN), and only alpha5beta1 is involved in cell adhesion to this glycoprotein, while alpha4beta1 mediates cell binding to the cellular counter-receptor VCAM-1. Proliferation assays showed that FN, but not VCAM-1, delivers a mitogenic signal to quiescent ADR(R) MCF-7 cells. The activating signal is mediated by alpha5beta1, since cell proliferation is inhibited in the presence of RGD peptide or specific antibody. Cell cycle analysis demonstrated that cell/FN interaction induces the re-entry of ADR(R) MCF-7 into S phase, and prevents them from undergoing serum deprivation-induced apoptosis. Our data suggest that the presence of alpha5beta1 on the resistant cells enables them to draw advantage from FN for both cell growth and survival.

Breast Neoplasms↗
PubMed1997

NKR-P1A stimulation of arachidonate-generating enzymes in rat NK cells is associated with granule release and cytotoxic activity.

NKR-P1A protein has been implicated in the triggering of NK-mediated natural killing contributing to target cell recognition by NK cells. The aim of the present work was to assess whether NKR-P1A receptor triggering also induced arachidonic acid (AA) generation and to determine the possible role of this event on granule release and cytotoxicity. We demonstrated that activation of fresh peripheral blood rat NK cells by cross-linking with the anti-NKR-P1A 3.2.3 mAb induced calcium-dependent AA release, which is due to the activation of cytosolic phospholipase A2 (cPLA2), secretory phospholipase A2 (sPLA2), and diacylglycerol/monoacylglycerol lipase. We also documented the presence of a type II sPLA2 activity in the supernatant fluids from NKR-P1A-activated rat NK cells, suggesting that AA and lysophospholipids could be mobilized from the outside of the cell. The involvement of AA-generating enzymes in NKR-P1A-induced cytotoxic functions was also investigated. Treatment of effector cells with arachidonyl trifluoromethylketone, a cPLA2 inhibitor; p-bromophenacylbromide, a sPLA2 inhibitor; or RHC80267, a diacylglycerol lipase inhibitor, led to a partial inhibition of the redirected lysis against P815 target cells as well the granule content release induced by NKR-P1A cross-linking. A complete abolishment of these events was observed when the cells were simultaneously incubated with all three inhibitors. Taken together, our results support a crucial role for the arachidonate-generating enzymes in the induction of lytic activity of NK cells directly or by leading to generation of additional mediators that can play a role in the context of NK cell activation and cytotoxic functions.

Animals↗
PubMed1997

Isolation and chromosomal localization of GPR31, a human gene encoding a putative G protein-coupled receptor.

The screening of a human genomic library with a chemokine receptor-like probe allowed us to obtain a putative member of the G protein-coupled receptor gene (GPCR) family, designated GPR31. Its deduced amino acid sequence encodes a polypeptide of 319 amino acids that shares 25-33% homology with members of the chemokine, purino, and somatostatin receptor gene families. Amino acid sequence comparison reveals that the best match in the protein databases is with the human orphan GPCR called HM74 (33% identity). Southern genomic analysis of the GPR31 gene shows a hybridization pattern consistent with that of a single-copy gene. Using fluorescence in situ hybridization, we have determined the chromosomal and regional localization of the GPR31 gene at 6q27. The GPR31 mRNA is expressed at low levels by several human cell lines of different cellular origins. The phylogenetic analysis suggests that the GPR31 receptor may represent a member of a new GPCR subfamily.

Amino Acid Sequence↗
PubMed1997

Expression of interleukin 15 (IL-15) in human rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma.

Interleukin 15 (IL-15) is a recently discovered cytokine that stimulates lymphocyte proliferation and migration via a trimeric receptor sharing the beta and gamma signal transducing chains with the IL-2 receptor. IL-15 is typically produced by normal cells that do not release IL-2, but little information is currently available on human tumors. To assess whether human musculo-skeletal sarcomas produce IL-15, we analyzed surgical specimens and cell lines obtained from rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma. IL-15 mRNA was present in 9/9 surgical specimens (3 Ewing's sarcomas, 5 osteosarcomas and 1 rhabdomyosarcoma). The analysis of a panel of cell lines (7 derived from Ewing's sarcoma, 12 from osteosarcoma and 5 from rhabdomyosarcoma) showed that all rhabdomyosarcoma and osteosarcoma cell lines expressed IL-15 mRNA at levels ranging from low to high, while Ewing's sarcoma cells contained little or no IL-15 message. ELISA assays showed IL-15 release in a subset of rhabdomyosarcomas and osteosarcomas, but not in Ewing's sarcoma. The highest production of IL-15, in the picogram/ml range, was found in rhabdomyosarcoma cell lines RH30 and RD.

Enzyme-Linked Immunosorbent Assay↗
PubMed1997

CD16 cross-linking induces both secretory and extracellular signal-regulated kinase (ERK)-dependent cytosolic phospholipase A2 (PLA2) activity in human natural killer cells: involvement of ERK, but not PLA2, in CD16-triggered granule exocytosis.

The phospholipase A2 (PLA2) enzymes play a central role in diverse cellular processes including phospholipid digestion and metabolism, host defense, and cell signaling. We investigated the ability of CD16 clustering to trigger PLA2 and extracellular signal-regulated kinase (ERK) activation in human NK cells, as well as their possible involvement in CD16-stimulated degranulation. Both secretory (sPLA2) and cytosolic (cPLA2) PLA2 were rapidly activated upon CD16 cross-linking; sPLA2 was found in the supernatant and also in a cell-associated form. cPLA2 activation was controlled by the ERK pathway as indicated by the close correlation between their kinetics of activation and by the ability of the specific MEK inhibitor, PD 098059, to abolish cPLA2 activation. CD16 stimulation also resulted in the generation of platelet-activating factor (PAF) and leukotrienes; both phospholipases contributed to their biosynthesis. Using the pharmacologic inhibitors AACOCF3, p-bromophenacyl bromide (pBPB), and PD 098059, which specifically inhibit cPLA2, sPLA2, and MEK, respectively, we demonstrated that the ERK signaling pathway, but not cytosolic or secretory PLA2, is required for CD16-triggered granule release.

Antibodies, Monoclonal↗
PubMed1997

Interleukin 1-beta-induced protein kinase C-zeta activation is mimicked by exogenous phospholipase D.

Interleukin 1-beta (IL1-beta) is a pleiotropic cytokine that stimulates a number of signal transduction pathways in cells, leading to different cellular responses. In this study we investigated the signal transduction pathways activated by IL1-beta in two different human cell lines: RD/TE671, a rhabdomyosarcoma, and EJ, a bladder-derived carcinoma. We showed that this cytokine induced the activation of protein kinase C-zeta (PKC-zeta) and the accumulation of a putative physiological PKC-zeta activator, phosphatidic acid [Limatola, Schaap, Moolenaar and van Blitterswijk (1994) Biochem. J. 304, 1001-1008]. Exogenously supplied phospholipase D, which generated cellular phosphatidic acid, was able to mimic the cytokine effect, supporting the hypothesis that this lipid second messenger might contribute to cytokine-induced PKC-zeta activation. In addition, we show that IL1-beta stimulation of BOSC23 cells, transiently overexpressing PKC-zeta, induced an increase in PKC-zeta autophosphorylation. These results give the first direct evidence that IL1-beta can activate this atypical PKC isoform and suggest that this enzyme might be involved in mediating some of the biological effects induced by IL1-beta.

Cell Line↗
PubMed1996

Retinoic acid-induced transcriptional modulation of the human interferon-gamma promoter.

Disregulation of vitamin A metabolism is able to generate different immunological effects, including altered response to infection, reduced IgG production, and differential regulation of cytokine gene expression (including interleukin-2 and -4 and interferon-gamma (IFN-gamma)). In particular, IFN-gamma gene expression is significantly affected by vitamin A and/or its derivatives (e.g. retinoic acid (RA)). Here, we analyze the effect of retinoic acid on IFN-gamma transcription. Transient transfection assays in the human T lymphoblastoid cell line Jurkat demonstrated that the activation of the IFN-gamma promoter was significantly down-regulated in the presence of RA. Surprisingly, two different AP-1/CREB-ATF-binding elements situated in the initial 108 base pairs of the IFN-gamma promoter and previously shown to be critical for transcriptional activity were unaffected by RA. Utilizing promoter deletions and electrophoretic mobility shift analysis, we identified a USF/EGR-1-binding element cooperating in the modulation of IFN-gamma promoter activity by RA. This element was found to be situated in a position of the IFN-gamma promoter close to a silencer element previously identified in our laboratory. These results suggest that direct modulation of IFN-gamma promoter activity is one of the possible mechanisms involved in the inhibitory effect of retinoids on IFN-gamma gene expression.

Humans↗
PubMed1996

Expression of transduced carcinoembryonic antigen gene in human rhabdomyosarcoma inhibits metastasis.

Carcinoembryonic antigen (CEA) is a highly glycosylated cell surface glycoprotein belonging to the immunoglobulin superfamily. CEA has been involved in vitro in adhesion mechanisms, but little is known about the function of this glycoprotein in vivo in normal tissue differentiation and malignancy. With regard to the relationship between CEA expression and tissue differentiation, it has been reported that transfection of the CEA gene in rat L6 myoblasts results in a complete block of myogenic differentiation. To extend investigations to the transformed myogenic counterpart and examine CEA effects on differentiation and malignancy outside the colon system, we have transfected the human CEA gene in human rhabdomyosarcoma cells. Human rhabdomyosarcoma cells transfected with the CEA gene correctly expressed membrane CEA anchored via glycosylphosphatidylinositol and secreted CEA in the medium. CEA gene transfer in human rhabdomyosarcoma cells, which display a limited differentiation ability, does not further inhibit myogenic differentiation or alter in vitro proliferation or natural killer sensitivity. CEA transfection does not affect s.c. growth in nude mice, but the ectopic expression of CEA in human rhabdomyosarcoma cells can strongly inhibit their metastatic ability to lungs and adrenals after i.v. injection. The impairment of metastatic potential correlates with a reduction in the homotypic adhesion properties of the cells. These data suggest that CEA, in some systems, can interfere with intercellular adhesion and, at least for cells not metastatic to the liver, can act as an anti-metastatic molecule.

Adrenal Gland Neoplasms↗
PubMed1996

Molecular cloning of TER1, a chemokine receptor-like gene expressed by lymphoid tissues.

Several chemokine receptors have been cloned and shown to belong to a superfamily of seven transmembrane, G protein-coupled receptors. We report here the molecular cloning of TER1, a novel human chemokine receptor-like gene. The amino acid sequence deduced from the TER1 cDNA shows 43, 40, 40, and 39% identity to CCR4, CCR5, CCR1, and CCR2B beta chemokine receptors, respectively. By the use of fluorescent in situ hybridization, we have mapped the TER1 gene to chromosome 3p21, clustered with other chemokine receptor genes. By Northern blot analysis, TER1 mRNA is found to be expressed in the thymus, spleen, and at barely detectable levels in peripheral blood lymphocytes. Moreover, TER1 message in abundant in the NK cell line NK3.3 and in the T cell line MOLT-4. The restricted TER1 expression in cells and tissues of the lymphoid lineage suggests that this receptor may play a role in regulating immune functions.

Amino Acid Sequence↗
PubMed1996

Phospholipase A2 activity and calpactin I levels in rat lymphokine-activated killer cells: correlation with the cytotoxic activity.

In the present paper we have shown evidence for a significant increase of type II sPLA2 activity in A-LAK cells. The A-LAK-mediated cytotoxicity against YAC-1 target cells was strongly inhibited by two inhibitors of sPLA2, p-BPB and mepacrine, suggesting the involvement of this enzyme in the lytic mechanism of A-LAK. On the other hand, stimuli such as A23187 ionophore and TPA, which were able to induce in control cells an increased AA release, failed to cause this effect in IL-2-treated cells, suggesting that PLA2 was not active in these cells. Thus, we analyzed the levels of calpactin I, which is considered to be involved in the down-regulation of PLA2 activity. HrIL-2 treatment led to an increased expression of calpactin I at both the RNA and the protein level. A substantial portion of calpactin I was associated with the external surface of A-LAK and was able to exert a strong inhibitory effect on a purified porcine pancreatic PLA2 activity in vitro. Our results suggest that the role of calpactin I could be relevant to regulate PLA2 activity, and to protect the effector cells against a possible toxic effect which this enzyme could exert if present at high levels.

Animals↗
PubMed1996

Cloning of a novel human RNA polymerase II subunit downregulated by doxorubicin: new potential mechanisms of drug related toxicity.

Using the differential display PCR method, we have isolated an mRNA downregulated in doxorubicin resistant human cell lines. The full length cDNA clone was identified as the human homologue of yeast RPB11 subunit of RNA polymerase II. Northern blot analysis of normal tissues detected a particularly high expression of RPB11 mRNA in heart and skeletal muscle. Reduction of this mRNA expression was observed in all the cell lines tested after drug treatment and was paralleled by a similar decrease of the protein levels. These findings suggest that doxorubicin may exert in vivo specific inhibitory effects on a major component of the transcription machinery.

Amino Acid Sequence↗
PubMed1996

Functional expression of Fas and Fas ligand on human gut lamina propria T lymphocytes. A potential role for the acidic sphingomyelinase pathway in normal immunoregulation.

The expression and function of Fas (CD95/APO-1), a cell surface receptor directly responsible for triggering cell death by apoptosis, was investigated on human T lymphocytes resident within the intestinal lamina propria, a major site of antigen challenge and persistent lymphocyte activation. Three color immunofluorescence and FACS analysis indicated that virtually all freshly isolated human gut lamina propria T lymphocytes (T-LPL) express Fas, together with the marker of progress activation CD45R0. A discrete fraction of freshly isolated T-LPL also constitutively expressed Fas ligand (FasL), perhaps as a result of recent in vivo activation. Importantly, whereas Fas cross-linking did not result in apoptosis induction in peripheral blood T lymphocytes (T-PBL), Fas was found to be fully effective in generating the apoptotic signal in T-LPL. This was associated with the activation of an acidic sphingomyelinase and with ceramide generation, early events known to be involved in Fas-mediated apoptotic signaling. By contrast, acidic sphingomyelinase activation and ceramide production were not detectable in T-PBL after Fas cross-linking. However C2-ceramide, a cell permeant synthetic analog of ceramide, could efficiently induce apoptosis in T-LPL and T-PBL when added exogenously. These data indicate that T-LPL constitutively express both Fas and FasL and that Fas cross-linking generates signals resulting in sphingomyelin hydrolysis and apoptosis, outlining a potential mechanism involved in intestinal tolerance. Moreover, they provide the first evidence of a role for ceramide-mediated pathways in normal immunoregulation.

Apoptosis↗
PubMed1996

CD16-mediated p21ras activation is associated with Shc and p36 tyrosine phosphorylation and their binding with Grb2 in human natural killer cells.

The Src homology (SH) 2/SH3 domain-containing protein Grb2 and the oncoprotein Shc have been implicated in a highly conserved mechanism that regulates p21ras activation. We investigated the involvement of these adaptor proteins in the signaling pathway induced by CD16 or interleukin (IL) 2R triggering in human natural killer (NK) cells. Both p46 and p52 forms of Shc were rapidly and transiently tyrosine phosphorylated upon CD16 or IL-2 stimulation with different kinetics. Shc immunoprecipitates from lysates of CD16- or IL-2-stimulated NK cells contained Grb2 and an unidentified 145-kD tyrosine phosphoprotein. Grb2 immunoprecipitates from anti-CD16-stimulated NK cells contained not only Shc, but also a 36-kD tyrosine phosphoprotein (p36). The interaction between Grb2 and Shc or p36 occurred via the Grb2SH2 domain as indicated by in vitro binding assays using a bacteriologically synthesized glutathione S-transferase-Grb2SH2 fusion protein. We also present evidence that p21ras is activated by CD16 and IL-2R cross-linking. Accumulation of guanosine triphosphate-bound Ras was detected within 1 minute and occurred with kinetics similar to inductive protein tyrosine phosphorylation and Grb2 association of Shc and p36 adaptor proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Functional analysis of alpha 1 beta 1 integrin in human natural killer cells.

Upon activation with interleukin (IL)-2 human natural killer (NK) cells acquire on their surface the alpha 1 beta 1 and alpha 2 beta 1 integrins and down-regulate the expression of alpha 6 beta 1. By employing alpha 1 beta 1-specific monoclonal antibody (mAb) HP-2B6, characterized in our laboratory, we examined the functional role of the alpha 1 beta 1 integrin in NK cells. Treatment with HP-2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti-alpha 2 beta 1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti-beta 1 LIA 1/2 mAb, but was unaffected by alpha 1 and alpha 2-specific mAb; as alpha 3 beta 1 and alpha 6 beta 1 were undetectable, the data indicate that the alpha 1 beta 1 integrin binding sites for type I collagen and laminin are different. Incubation with anti-alpha 1 HP-2B6 or its F(ab')2 fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca2+ and Mg2+); homotypic cell adhesion was selectively blocked by anti-CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti-alpha 1 HP-2B6 enhanced TNF-alpha production and induced tyrosine phosphorylation of a 110-kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti-alpha 1 HP-2B6 mAb. Our data show that ligation of the alpha 1 beta 1 integrin positively modulates IL-2-activated NK cell function via a PTK-dependent pathway.

Animals↗
PubMed1996

Tyrosine kinase-dependent activation of human NK cell functions upon triggering through CD44 receptor.

We recently showed evidence of CD44-mediated enhancement of natural killer (NK) cell cytotoxic activity and induction of intracellular Ca2+ flux. In this study, we evaluated whether CD44 plays a stimulatory role in NK cell functions, such as cytokine production and activation antigen expression. Our results indicate that ligation of the CD44 receptor results in the induction of expression of the CD69 surface activation antigen as well as in the enhancement of phorbol ester-induced TNF-alpha secretion. We report also evidence for the coupling of CD44 receptor to a protein tyrosine kinase(s) pathway. CD44 engagement rapidly stimulates the tyrosine phosphorylation of several cellular substrates. Pretreatment of NK cells with the tyrosine kinase inhibitor herbimycin A resulted in marked decrease of CD44-stimulated phosphorylation, indicating that it activates tyrosine kinase(s). Furthermore, the drug also prevents CD44-mediated TNF-alpha production and CD69 expression. These findings indicate that protein tyrosine phosphorylation is an early and critical event in CD44-mediated activation of NK cell functions.

Antigens, CD↗