Biomedical subjects

B Robaye

Publications and source records attributed to B Robaye.

At least 19 recordsLinked to original sources

PubMed2001

The P2Y11 receptor mediates the ATP-induced maturation of human monocyte-derived dendritic cells.

Recently, it has been shown that ATP and TNF-alpha synergize in the activation and maturation of human dendritic cells (DC); the effect of ATP was reproduced by hydrolysis-resistant derivatives of ATP and was blocked by suramin, suggesting the involvement of a P2 receptor, but the particular subtype involved was not identified. In this report we confirm that ATP and various derivatives synergize with TNF-alpha and LPS to induce the maturation of human monocyte-derived DC, as revealed by up-regulation of the CD83 marker and the secretion of IL-12. The rank order of potency of various analogs (AR-C67085 > adenosine 5'-O-(3-thiotriphosphate) = 2'- and 3'-O-(4-benzoyl-benzoyl) ATP > ATP > 2-methylthio-ATP) was close to that of the recombinant human P2Y11 receptor. Furthermore, these compounds activated cAMP production in DC, in a xanthine-insensitive way, consistent with the involvement of the P2Y11 receptor, which among P2Y subtypes has the unique feature of being dually coupled to phospholipase C and adenylyl cyclase activation. The involvement of the P2Y11/cAMP/protein kinase A signaling pathway in the nucleotide-induced maturation of DC is supported by the inhibitory effect of H89, a protein kinase A inhibitor. Taken together, our results demonstrate that ATP activates DC through stimulation of the P2Y11 receptor and subsequent increase in intracellular cAMP.

Adenosine Triphosphate↗

PubMed2001

Molecular cloning and characterization of the mouse P2Y4 nucleotide receptor.

To isolate the mouse P2Y4 receptor gene, a mouse genomic library was screened with a human P2Y4 probe. An open reading frame encoding a protein of 361 amino acids was isolated. This protein showed 82% and 95% amino acid identity with the human and rat P2Y4 receptors, respectively. By reverse transcription and polymerase chain reaction (RT-PCR), the P2Y4 messenger RNA was detected in mouse liver, intestine, stomach, bladder and lung among the 16 mouse tissues tested. In 1321N1 transfected cells, the mouse P2Y4 receptor was equally activated by UTP and ATP, and was antagonized by pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and Reactive Blue 2, and not by suramin. Moreover, when expressed in 1321N1 cells, the rat P2Y4 is also antagonized by PPADS. Thus, when compared in the same expression system, the mouse P2Y4 is closer to the rat ortholog in terms of agonist stimulation, while in terms of antagonist profile, the three P2Y4 receptor orthologs are similar.

Adenosine Triphosphate↗

PubMed2001

Perforin and granzyme B induce apoptosis in FasL-resistant colon carcinoma cells.

Cytotoxic lymphocytes may induce apoptosis in their target cells by the FasL (Fas ligand) pathway or the perforin/granzyme B pathway. It has been shown that Fas-expressing colon carcinoma (CC) cells are resistant to FasL-mediated apoptosis. The aims of this study were to determine whether CC cells are also resistant to perforin/granzyme B and whether the FasL resistance lies upstream of caspase-3 activation. The resistance of the Fas-expressing rat CC531s cells to the FasL pathway was confirmed by treating them with recombinant human soluble FasL, using rat hepatocytes as a positive control. The intracellular delivery of granzyme B by sublytic concentrations of perforin, on the other hand, resulted in many features of apoptosis (chromatin condensation, nucleus fragmentation, loss of microvilli and internucleosomal DNA fragmentation) within 3 h. Since both the FasL and perforin/granzyme B pathways converge at caspase-3, we measured caspase-3 activity to learn whether the FasL resistance was due to failure to activate this crucial executioner. Caspase-3 activation occurred in CC531s cells after perforin/granzyme B treatment, but not after the addition of recombinant FasL. Furthermore, we showed that caspase-3 activity is involved in the execution of perforin/granzyme-B-induced apoptosis in CC531 s cells, since the cell-permeable caspase-3 inhibitor Z-DEVD-FMK abrogated DNA fragmentation. Together, these results suggest that CC cells are sensitive to perforin/granzyme-B-induced apoptosis by activating caspase-3 and FasL resistance lies upstream of this executioner caspase.

Apoptosis↗

PubMed2001

Low TSH requirement and goiter in transgenic mice overexpressing IGF-I and IGF-Ir receptor in the thyroid gland.

Through the cAMP signaling pathway, TSH stimulates thyroid follicular cell proliferation, differentiation, and function. Although the autocrine production of IGF-I in the thyroid gland suggests an important physiological function for this factor in these processes, the exact role of the IGF-I/IGF-I receptor system in vivo remains unclear. Although the mitogenic action of TSH requires the presence of IGF-I or insulin in primary culture of dog and human thyroid cells, IGF-I has an effect equal to and independent of the effect of TSH on cell proliferation in rat thyroid cell lines and may even be the main growth regulator in this case. To investigate the in vivo function of the IGF-I/IGF-I receptor system, transgenic mice overexpressing human IGF-I, IGF-I receptor, or both in the thyroid were generated. Adult transgenic mice did not present external signs of thyroid dysfunction, but mice overexpressing both transgenes had significantly increased gland weight and follicular lumen area. A decreased TSH level together with a slightly increased serum T(4) concentration and increased thyroidal iodine uptake were also observed, suggesting that IGF-I and IGF-I receptor stimulate thyroid function to some extent in vivo.

Animals↗

PubMed2000

Rapid up-regulation of P2Y messengers during granulocytic differentiation of HL-60 cells.

HL-60 cells are human promyelocytic cells expressing two ATP receptors: the P2Y(2) and P2Y(11) subtypes. Our Northern blotting experiments have shown that P2Y(2) and P2Y(11) messengers were up-regulated in these cells, rapidly and independently of protein synthesis, following treatment with granulocytic differentiating agents such as retinoic acid, dimethylsulfoxide, granulocyte-colony stimulating factor, dibutyryl cyclic AMP and ATP. AR-C67085 and adenosine 5'-O-(3-thiotriphosphate), two potent agonists of the recombinant P2Y(11) receptor, increased intracellular cAMP concentration in HL-60 cells more potently than ATP itself. These observations support the conclusion that the effect of ATP on HL-60 cell differentiation is mediated by the P2Y(11) receptor.

Cell Differentiation↗

PubMed2000

Actin depolymerization and polymerization are required during apoptosis in endothelial cells.

In order to understand the role of actin microfilaments in the apoptotic process, we followed their evolution during tumor necrosis factor-alpha (TNF)-induced apoptosis in bovine aortic endothelial (BAE) cells. Using Western blotting analysis and immunofluorescence microscopy, we observed that the actin microfilaments network was disrupted in apoptotic cells. Depolymerization of F-actin was concomitant with internucleosomal DNA degradation and with the morphological changes associated with apoptotic cell death. However, using the actin microfilament disrupting agent, cytochalasin, we present evidence that the formation of blebs leading to apoptotic cell fragmentation requires neopolymerization of actin. Indeed, in the presence of cyochalasin, induction of apoptosis (internucleosomal DNA degradation) in BAE cells by TNF and cycloheximide was not associated with these classical morphological markers of apoptosis. Moreover, when added to BAE cells showing incipient apoptotic fragmentation, cytochalasin E reversed this process. We also observed an accumulation of actin at the basis of the apoptotic bodies in formation in these cells. Together, these results suggest that the actin network of flattened cells is disrupted concomitantly to the morphological modifications associated to the apoptotic cell death, and that the cytochalasin-sensitive reorganisation of actin is required to the formation of apoptotic blebs.

Actins↗

PubMed2000

Advances in signalling by extracellular nucleotides. the role and transduction mechanisms of P2Y receptors.

Nucleotides are ubiquitous intercellular messengers whose actions are mediated by specific receptors. Since the first clonings in 1993, it is known that nucleotide receptors belong to two families: the ionotropic P2X receptors and the metabotropic P2Y receptors. Five human P2Y receptor subtypes have been cloned so far and a sixth one must still be isolated. In this review we will show that they differ by their preference for adenine versus uracil nucleotides and triphospho versus diphospho nucleotides, as well as by their transduction mechanisms and cell expression.

Adenylyl Cyclases↗

PubMed2000

Myosin heavy chain degradation during apoptosis in endothelial cells.

The cytoskeleton undergoes dramatic changes during apoptosis and many cytoskeletal proteins are known to be degraded during this process. The number of proteases found to be involved in apoptosis is growing but the role of the proteolysis they cause remains poorly understood. This report describes for the first time that myosin heavy chain is cleaved in aortic endothelial cell apoptosis induced either by tumour necrosis factor-alpha or okadaic acid. The cleavage was specific since a well-defined major 97 kDa fragment of myosin heavy chain was produced. The intermediate filament component vimentin was also cleaved into well-defined fragments (31, 28 and 23 kDa). Kinetic studies showed that proteolysis occurred concomitantly with the morphological changes associated with apoptosis, i.e. cellular condensation and fragmentation in apoptotic bodies. These data suggest that the degradation of myosin and vimentin could be involved in the execution of the morphological alterations observed during apoptotic cell death.

Amino Acid Chloromethyl Ketones↗

PubMed1999

cAMP induces co-translational modification of proteins in IPC-81 cells.

An elevated cAMP concentration results in growth arrest and protein synthesis-dependent apoptosis in the promyelocytic leukaemia cell line IPC-81. A comparison of two-dimensional gels of extracts from these cells labelled with [(35)S]methionine revealed that five distinct protein spots were induced by cAMP in a protein-synthesis-dependent manner. The spots seemed to result from the acidic shift of a precursor protein. The most abundant spot was phospho-actin. The spots induced by cAMP in intact cells were induced by cAMP-dependent protein kinase (cAPK) during the translation in vitro of mRNA from the leukaemia cells. The effect of cAPK was strictly co-translational, none of the spots being induced when cAPK was added after translation. This suggested that the protein spots arose by co-translational phosphorylation catalysed by cAPK. Two of the protein spots, phospho-actin and a protein with a molecular mass of 30 kDa and an isoelectric point of 4.5, were studied further with respect to expression. They were produced during the whole pre-apoptotic period, had cellular half-lives of several hours and were induced by the same concentrations of cAMP analogue that induced apoptosis. It is suggested that the accumulation of co-translationally modified proteins could be important for long-term cAMP signalling.

Actins↗

PubMed1999

Pit cells (Hepatic natural killer cells) of the rat induce apoptosis in colon carcinoma cells by the perforin/granzyme pathway.

The high mortality of colon cancer is to a large extent caused by the frequent occurrence of liver metastasis. This is remarkable, because the liver harbors two distinct cell populations that can eliminate invading cancer cells, namely hepatic natural killer (NK) cells and Kupffer cells. These hepatic NK cells, known as pit cells, are the most cytotoxic cells of the naturally occurring NK cells. However, the mechanism by which pit cells eliminate tumor cells is largely unknown. Because we recently found an indication that apoptosis is involved, we tried to assess the role of this mode of cell death using an in vitro system with isolated pure pit cells (>90%) and CC531s cells, a rat colon carcinoma (CC) cell line. Pit cells induced apoptosis in CC531s cells as shown by quantitative DNA fragmentation, agarose gel electrophoresis, and different modes of microscopy. When extracellular Ca2+ was chelated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,-tetraacetic acid (EGTA) during coincubation or when the pit cells were preincubated with the granzyme inhibitor 3,4-dichloroisocoumarin (DCI), the induction of apoptosis was abolished. These results show that pit cells use the Ca2+-dependent perforin/granzyme pathway to induce apoptosis in the CC531s cells, and not the alternative Ca2+-independent Fas pathway. To further exclude the possibility of the involvement of the Fas pathway, we treated CC531s cells with recombinant Fas ligand. This treatment did not result in the induction of apoptosis, indicating that CC531s cells are resistant to Fas-mediated apoptosis. We conclude therefore that pit cells induce apoptosis in CC cells in vitro by the perforin/granzyme pathway.

Animals↗

PubMed1999

Pharmacological characterization of the human P2Y11 receptor.

1 The human P2Y11 receptor is coupled to both the phosphoinositide and the cyclic AMP pathways. A pharmacological characterization of the recombinant human P2Y11 receptor has been conducted following stable expression in two different cell lines: the 1321N1 astrocytoma cells for inositol trisphosphate measurements and the CHO-K1 cells for cyclic AMP assays. The rank order of potency of a series of nucleotides was almost identical for the two pathways: ATPgammaS approximately BzATP > dATP > ATP > ADPbetaS > 2MeSATP. 2 ADPbetaS, AMPalphaS and A3P5PS behaved as partial agonists of the human P2Y11 receptor. At high concentrations, these three nucleotides were able to partially inhibit the ATP response. 3 Suramin was a more potent antagonist than reactive blue 2, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid was completely inactive. The P2Y11 receptor proved to be sensitive to suramin in a competitive way with an apparent Ki value of 0.82+/-0. 07 microM. 4 The ATP derivative AR-C67085 (2-propylthio-beta, gamma-dichloromethylene-D-ATP), a potent inhibitor of ADP-induced platelet aggregation, was the most potent agonist of the P2Y11 receptor, among the various nucleotides tested. 5 The pharmacological profile of the recombinant human P2Y11 receptor is closely similar to that of the cyclic AMP-coupled P2 receptor recently described in HL-60 cells, suggesting that it is the same receptor.

Adenosine Diphosphate↗

PubMed1998

Expression of HSP27 results in increased sensitivity to tumor necrosis factor, etoposide, and H2O2 in an oxidative stress-resistant cell line.

The role of HSP27 in cell growth and resistance to stress was investigated using murine fibrosarcoma L929 cells (normally devoid of constitutively expressed small HSPs) and human osteoblast-like SaOS-2 cells stably transfected with a human hsp27 expression vector. Our data showed that our L929 cells were more resistant to oxidative stress than generally observed for this line. Production of HSP27 in these cells led to a marked decrease in growth rate associated with a series of phenotypical changes, including cell spreading, cellular and nuclear hypertrophy, development of an irregular outline, and a tremendous accumulation of actin stress fibers. By contrast, none of these changes was observable in SaOS-2/hsp27 transfectants overexpressing the protein product. Together, these observations are consistent with a cause-to-effect cascade relationship between increased (or induced) HSP27 expression, changes in cytoskeletal organization, and decreased growth. On the other hand, whereas the transfection of the hsp27 gene increased the cell resistance to heat in both cell lines, only in SaOS-2 cells was this associated with protection to the cytotoxic action of tumor necrosis factor-alpha (TNF-alpha) and etoposide. Unexpectedly, L929/hsp27 transfectants exhibited an increased sensitivity to both agents and also to H2O2. These data thus imply that different mechanisms are involved in the cell resistance to heat shock and to the cytotoxic action of TNF-alpha, etoposide, and H2O2. They also plead against the simple view that overexpression of a phosphorylatable HSP27 would necessarily be beneficial in terms of increased cell resistance to any type of stress. Our data further indicate that the role of HSP27 in cellular resistance to stress and in cell proliferation involves different targets and that the ultimate result of its interference with these processes depends on the intracellular context in which the protein is expressed.

Actins↗

PubMed1997

Characterization of a phosphoprotein whose mRNA is regulated by the mitogenic pathways in dog thyroid cells.

We have isolated cDNA clones encoding the dog and human forms of a novel protein whose function is still unknown. Sequence analysis indicates that dog clone c5fw protein contains 343 amino acid residues. several potential phosphorylation sites. and two of the 12 conserved subdomains (VIII and IX) that fold into a common catalytic core structure of the large family of protein kinases. Human clone c5fw shares 95% amino acid identity with its dog counterpart. We have also isolated another human-related clone c5fw sharing 70% amino acid identity with the dog sequence. We transiently expressed c-myc epitope-tagged clone c5fw protein in COS-7 cells and infected thyrocytes in primary culture with a recombinant adenovirus containing clone c5fw cDNA (adenovirus c5fw). In both experiments, a 46-kDa protein was detected and subsequently more extensively characterized. By two-dimensional gel electrophoresis and V8 protease digestion, we showed that this overexpressed protein is phosphorylated on different sites. Moreover, cells stimulated with thyrotropin or epidermal growth factor, thyrotropin and fetal calf serum increased the level of clone c5fw protein produced after infection by adenovirus containing clone c5fw. The disappearance of this 46-kDa protein after 1 h of puromycin treatment indicates that it is a labile protein. Immunofluorescence and subcellular fractionation analysis have revealed that c-myc-tagged clone c5fw was insoluble and localized mainly in the cytoplasm, in the form of granules.

Adenoviridae↗

PubMed1997

Slow desensitization of the human P2Y6 receptor.

The P2Y6 receptor is a recently cloned P2 receptor which displays a high sensitivity for diphosphonucleotides. In 1321N1 astrocytoma cells stably expressing this receptor, UDP induced a slow and sustained accumulation of inositol trisphosphate via a pertussis toxin-insensitive G-protein: the maximal level was only reached after 15 min and a significant response was maintained for at least 3 h. A full second response to UDP was obtained after the first 45-min stimulation, but was lost after 165 min. This slow and sustained time-course and the lack of desensitization was reproduced with ADP. UTP was unable to restimulate the P2Y4 receptor, another recently cloned P2 receptor with a preference for UTP, after the first 5-min stimulation. The P2Y4 receptor is thus rapidly desensitized whereas desensitization of the P2Y6 receptor is delayed. The rank order of potency of various diphosphonucleotides at the P2Y6 receptor was: UDP > TDP > IDP > GDP > ADP >> CDP. The activity of three non-specific antagonists of P2 receptors was characterized by the following rank order of potency: reactive blue 2 > pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) > suramin. In conclusion, the most impressive features of the human P2Y6 receptor revealed by this study are the slow and sustained time-course of its activation and its high resistance to desensitization.

Adenosine Diphosphate↗

PubMed1996

Action of cAMP on expression and release of adhesion molecules in human endothelial cells.

The expression of E-selectin induced by tumor necrosis factor (TNF) on the surface of human umbilical vein endothelial cells (HUVEC) was partially inhibited by an increase in the level of adenosine 3',5'-cyclic monophosphate (cAMP), produced by forskolin or cholera toxin combined with the type IV phosphodiesterase inhibitor rolipram and the protein kinase A agonist phosphorothioate analogue of cAMP SpcAMPS. The same agents had no significant effect on the constitutive and TNF-stimulated expression of intercellular adhesion molecule 1 (ICAM-1), whereas the effect on vascular cell adhesion molecule 1 (VCAM-1) expression was variable depending on cell culture conditions. The stimulatory effects of phorbol 12-myristate 13-acetate and bacterial lipopolysaccharide (LPS) on E-selectin expression were also downregulated by the forskolin-rolipram combination and by SpcAMPS. Inhibition of the surface expression of E-selectin was associated with a decrease of the total amount of the protein in the cell lysate and a reduced mRNA level, with no significant effect on mRNA stability. In anesthetized rats, the terbutaline-rolipram combination reduced the rolling of leukocytes induced by LPS in the mesenteric microcirculation. In addition to their partial inhibitory effect on the TNF-induced surface expression of E-selectin on HUVEC, the forskolin-rolipram combination and SpcAMPS strongly inhibited the release of soluble E-selectin from these cells; the release of soluble ICAM-1 and VCAM-1 was unaffected by these agents. Isoproterenol reduced the release of soluble E-selectin, whereas it had no significant effect on the cell surface expression of the protein. This study underscores the potential anti-inflammatory effect of a rise in the endothelial cAMP level.

Animals↗

PubMed1995

Fine mapping of 28S rRNA sites specifically cleaved in cells undergoing apoptosis.

Bona fide apoptosis in rat and human leukemia cells, rat thymocytes, and bovine endothelial cells was accompanied by limited and specific cleavage of polysome-associated and monosome-associated 28S rRNA, with 18S rRNA being spared. Specific 28S rRNA cleavage was observed in all instances of apoptotic death accompanied by internucleosomal DNA fragmentation, with cleavage of 28S rRNA and of DNA being linked temporally. This indicates that 28S rRNA fragmentation may be as general a feature of apoptosis as internucleosomal DNA fragmentation and that concerted specific cleavage of intra- and extranuclear polynucleotides occurs in apoptosis. Apoptosis-associated cleavage sites were mapped to the 28S rRNA divergent domains D2, D6 (endothelial cells), and D8. The D2 cuts occurred in hairpin loop junctions considered to be buried in the intact ribosome, suggesting that this rRNA region becomes a target for RNase attack in apoptotic cells. D8 was cleaved in two exposed UU(U) sequences in bulge loops. Treatment with agents causing necrotic cell death or aging of cell lysates failed to produce any detectable limited D2 cleavage but did produce a more generalized cleavage in the D8 region. Of potential functional interest was the finding that the primary cuts in D2 exactly flanked a 0.3-kb hypervariable subdomain (D2c), allowing excision of the latter. The implication of hypervariable rRNA domains in apoptosis represents the first association of any functional process with these enigmatic parts of the ribosomes.

Animals↗

PubMed1994

Apoptosis in dog thyroid cells.

Apoptosis, a physiological cell death, has been shown to be involved in tissue homeostasis as well as in tissue regression due to hormone deprivation. The cell population slowly turns over, with cell loss compensating mitogenicity. In the absence of thyrotropin, the cell population decreases. The possible involvement of apoptosis in this loss has been studied. We show in this report that deprivation of serum, epidermal growth factor and thyrotropin triggers internucleosomal DNA fragmentation and morphological modifications characteristic of apoptotic cell death in dog thyroid cells in primary culture; cycloheximide treatment has the same effect. This indicates that thyrocytes are endowed with a constitutive apoptosis "program" and that the latter might be involved in the thyroid regression observed in vivo in the absence of full growth stimulation of the gland.

Animals↗

PubMed1993

A model for tumor suppression using H-1 parvovirus.

A model system is proposed to investigate, at the molecular level, the pathways of tumor suppression. As a tool for the selection of cells with a suppressed phenotype, we used the H-1 parvovirus that preferentially kills various neoplastic cells. From the human K562 leukemia cells, we isolated a clone, KS, that is resistant to the cytopathic effect of the H-1 virus and displays a suppressed malignant phenotype. The suppressed malignancy and the cellular resistance to H-1 killing appear to depend on the activity of wild-type p53. Whereas the KS cells express wild-type p53, the protein is undetectable in the parental K562 cells. Experiments with p53 mutants suggest that wild-type p53, in its functionally intact state, contributes to the resistance against the cytopathic effect of H-1 parvovirus.

Antigens, CD↗