Calcium mobilisation controls tyrosine protein phosphorylation independently of the activation of protein kinase C in human platelets

AbstractWe have investigated the regulation of tyrosine proteins phosphorylation by intracellular Ca2+ level ([Ca2+]i) and protein kinase C (PKC) during platelet stimulation. We found that chelation of extracellular calcium completely prevented phosphorylation of tyrosine proteins induced by thapsigargin and phorbol 12-myristate 13-acetate (PMA), whereas, when induced by thrombin, it prevented a subset of tyrosine proteins. The selective inhibition of PKC by OF 109203X did not abolish tyrosine protein phosphorylation when induced by thrombin and thapsigargin. The results suggest that in human platelets tyrosine protein phosphorylation is dependent on [Ca2+]i, although direct PKC activation can also induce phosphorylation of tyrosine proteins

Allium compounds, dipropyl and dimethyl thiosulfinates, as antiproliferative and differentiating agents of human acute myeloid leukemia cell lines: Antiproliferative effects of thiosulfinates

There is increasing evidence that certain Allium derivatives have beneficial effects on coronary diseases and cancer. Thus, thiosulfinates have already been shown to inhibit platelet aggregation and clot retraction. Here, we have examined the effects of dipropyl and dimethyl thiosulfinates against acute myeloid leukemia (AML) cell lines. Both thiosulfinates inhibited proliferation of cell lines in a concentation-dependent fashion without inducing necrosis. Moreover, they inhibited the expression of matrix metalloproteinase-9 (MMP-9) protein and its gelatinolytic activity. The mechanisms by which these molecules inhibit MMP9 are now studied using the RT-PCR approach. In parallel, the effects of the related molecules dipropyl and dimethyldisulfides are being evaluated. Already, our data highlight the potential application of such molecules to cancer control.International audienceEpidemiologic studies support the premise that Allium vegetables may lower the risk of cancers. The beneficial effects appear related to the organosulfur products generated upon processing of Allium. Leukemia cells from patients with acute myeloid leukemia (AML) display high proliferative capacity and have a reduced capacity of undergoing apoptosis and maturation. Whether the sulfur-containing molecules thiosulfinates (TS), diallyl TS (All2TS), dipropyl TS (Pr2TS) and dimethyl TS (Me2TS), are able to exert chemopreventative activity against AML is presently unknown. The present study was an evaluation of proliferation, cytotoxicity, differentiation and secretion of AML cell lines (U937, NB4, HL-60, MonoMac-6) in response to treatment with these TS and their related sulfides (diallylsulfide, diallyl disulfide, dipropyl disulfide, dimethyl disulfide). As assessed by flow cytometry, ELISA, gelatin zymogaphy and RT-PCR, we showed that Pr2TS and Me2TS, but not All2TS and sulfides, 1) inhibited cell proliferation in dose- and time-dependent manner and this process was neither due to cytotoxicity nor apoptosis, 2) induced macrophage maturation, and 3) inhibited the levels of secreted MMP-9 (protein and activity) and TNF-? protein, without altering mRNA levels. By establishing for the first time that Pr2TS and Me2TS affect proliferation, differentiation and secretion of leukemic cel lines, this study provides the opportunity to explore the potential efficiency of these molecules in AML

Allium compounds, dipropyl and dimethyl thiosulfinates as antiproliferative and differentiating agents of human acute myeloid leukemia cell lines

Epidemiologic studies support the premise that Allium vegetables may lower the risk of cancers. The beneficial effects appear related to the organosulfur products generated upon processing of Allium. Leukemia cells from patients with acute myeloid leukemia (AML) display high proliferative capacity and have a reduced capacity of undergoing apoptosis and maturation. Whether the sulfur-containing molecules thiosulfinates (TS), diallyl TS (All2TS), dipropyl TS (Pr2TS) and dimethyl TS (Me2TS), are able to exert chemopreventative activity against AML is presently unknown. The present study was an evaluation of proliferation, cytotoxicity, differentiation and secretion of AML cell lines (U937, NB4, HL-60, MonoMac-6) in response to treatment with these TS and their related sulfides (diallylsulfide, diallyl disulfide, dipropyl disulfide, dimethyl disulfide). As assessed by flow cytometry, ELISA, gelatin zymogaphy and RT-PCR, we showed that Pr2TS and Me2TS, but not All2TS and sulfides, 1) inhibited cell proliferation in dose- and time-dependent manner and this process was neither due to cytotoxicity nor apoptosis, 2) induced macrophage maturation, and 3) inhibited the levels of secreted MMP-9 (protein and activity) and TNF-α protein, without altering mRNA levels. By establishing for the first time that Pr2TS and Me2TS affect proliferation, differentiation and secretion of leukemic cell lines, this study provides the opportunity to explore the potential efficiency of these molecules in AML

Smoking Related Diseases: The Central Role of Monoamine Oxidase

Smoking is a major risk factor of morbidity and mortality. It is well established that monoamine oxidase (MAO) activity is decreased in smokers. Serotonin (5-HT), a major substrate for MAO that circulates as a reserve pool stored in platelets, is a marker of platelet activation. We recently reported that smoking durably modifies the platelet 5-HT/MAO system by inducing a demethylation of the MAO gene promoter resulting in high MAO protein concentration persisting more than ten years after quitting smoking. The present data enlarges the results to another MAO substrate, norepinephrine (NE), further confirming the central role of MAO in tobacco use-induced diseases. Thus, MAO could be a readily accessible and helpful marker in the risk evaluation of smoking-related diseases, from cardiovascular and pulmonary diseases to depression, anxiety and cancer. The present review implements the new finding of epigenetic regulation of MAO and suggests that smoking-induced MAO demethylation can be considered as a hallmark of smoking-related cancers similarly to other aberrant DNA methylations

Smoking-induced long-lasting modifications of human platelet serotonin catabolism through a MAO epigenetic regulation

Postulating that serotonin, secreted from smoking-activated platelets, could be involved in smoking-induced vascular modifications, we studied 115 men distributed in smokers (S), former smokers (FS) and never smokers (NS). The platelet serotonin content was similar in S and NS but lower in FS. This was unexpected because the monoamine oxidase (MAO) activity, which catabolizes serotonin, was inhibited during smoking. However, the amount of platelet MAO was higher in S and FS than in NS. The persistent elevated MAO amount in FS prompted us to study the methylation of its gene promoter in an additional series of patients: it was markedly lower for S and FS vs. NS due to cigarette smoke-induced increase of nucleic acid demethylase activity. This smoking-induced demethylation of the MAO gene promoter, resulting in high MAO amount persisting long after quitting smoking, has cardiovascular consequences and could impact fields such as behavior, mental health, and cancer

Smoking Induces Long-Lasting Effects through a Monoamine-Oxidase Epigenetic Regulation

BACKGROUND: Postulating that serotonin (5-HT), released from smoking-activated platelets could be involved in smoking-induced vascular modifications, we studied its catabolism in a series of 115 men distributed as current smokers (S), never smokers (NS) and former smokers (FS) who had stopped smoking for a mean of 13 years. METHODOLOGY/PRINCIPAL FINDINGS: 5-HT, monoamine oxidase (MAO-B) activities and amounts were measured in platelets, and 5-hydroxyindolacetic acid (5-HIAA)--the 5-HT/MAO catabolite--in plasma samples. Both platelet 5-HT and plasma 5-HIAA levels were correlated with the 10-year cardiovascular Framingham relative risk (P<0.01), but these correlations became non-significant after adjustment for smoking status, underlining that the determining risk factor among those taken into account in the Framingham risk calculation was smoking. Surprisingly, the platelet 5-HT content was similar in S and NS but lower in FS with a parallel higher plasma level of 5-HIAA in FS. This was unforeseen since MAO-B activity was inhibited during smoking (P<0.00001). It was, however, consistent with a higher enzyme protein concentration found in S and FS than in NS (P<0.001). It thus appears that MAO inhibition during smoking was compensated by a higher synthesis. To investigate the persistent increase in MAO-B protein concentration, a study of the methylation of its gene promoter was undertaken in a small supplementary cohort of similar subjects. We found that the methylation frequency of the MAOB gene promoter was markedly lower (P<0.0001) for S and FS vs. NS due to cigarette smoke-induced increase of nucleic acid demethylase activity. CONCLUSIONS/SIGNIFICANCE: This is one of the first reports that smoking induces an epigenetic modification. A better understanding of the epigenome may help to further elucidate the physiopathology and the development of new therapeutic approaches to tobacco addiction. The results could have a larger impact than cardiovascular damage, considering that MAO-dependent 5-HT catabolism is also involved in addiction, predisposition to cancer, behaviour and mental health

Etude de la signalisation plaquettaire (implication de p120Cbl, phosphatidylinositol 3-kinase (PI 3-K) et Grb2)

Les plaquettes sanguines sont des cellules différenciées provenant de la fragmentation des mégacaryocytes. Annucléées et donc incapables de proliférer, les plaquettes réagissent aux signaux activateurs par des réponses du type adhésion, agrégation et une activité procoagulante. Les réponses plaquettaires diffèrent selon l'agoniste et la voie de transduction du signal activée. L'action d'un agoniste sur les plaquettes transmet un signal, dit inside-out, activateur de l'intégrine aIIb-b3. En conséquence, aIIb-b3 activée fixe le fibrinogène et permet la transmission d'un signal activateur secondaire, dit outside-in. La première partie de ces travaux a consisté en l'étude de l'implication de la protéine adaptatrice Cbl et de la lipide kinase, PI 3-K, dans les voies de signalisation médiées par (i) le récepteur du domaine Fc des IgG (FcgRIIa), (ii) l'intégrine aIIb-b3 (outside-in) et le récepteur de la thrombine. Nous avons montré que Cbl est fortement phosphorylée sur tyrosine au cours de l'activation de la voie de (FcgRIIa), alors qu'elle ne l'est que faiblement à la thrombine...Blood platelets are anucleated and terminaly differenciated cells. A number of receptors are present on platelet membrane and mediate signalling and activation in response to various agonists. The action of an agonist triggers an inside-out signalling which activates aIIb-b3 integrin. The latter becomes able to link fibrinogen and transduces a secondary outside-in signalling. We first studied the involvement of the adoptor protein, Cbl, and of the lipid kinase, PI 3-K, in platelet signalling mediated by (i) the receptor for Fc domain of IgG (FcgRIIa), (ii) by the aIIb-b3 integrin (outside-in) and (iii) by the thrombin receptor...PARIS-BIUP (751062107) / SudocSudocFranceF

Développement d'une nouvelle méthode de déshydratation des plaquettes sanguines par zéodratation

Les plaquettes sanguines sont conservées dans les centres de transfusion pendant 5 jours, à température ambiante et sous agitation constante. Ces conditions ne permettent pas leur stockage à long terme ni leur transport, pourtant nécessaire afin de répondre aux besoins de la médecine de catastrophe. Les méthodes de stockage à long terme développées jusqu ici n ont pas apporté satisfaction puisque l exposition au froid altère la recirculation et la fonctionnalité des plaquettes. Dans ce contexte, nous avons adapté un procédé innovant de déshydratation à température ambiante : la zéodratation. Les objectifs de cette thèse ont été (i) de concevoir un zéodrateur à l échelle pilote, (ii) d identifier les conditions de zéodratation adaptées au stockage des plaquettes sanguines, (iii) de caractériser les plaquettes zéodratées/réhydratées in vitro (iv) puis d évaluer leur capacité hémostatique in vivo. Cette étude a mis en évidence la faisabilité de la méthode de zéodratation pour le stockage à long terme des plaquettes. Malgré leur morphologie altérée, les plaquettes zéodratées sont capables de corriger l hémostase dans un modèle murin de saignement. Des recherches concernant l amélioration de la fonctionnalité des plaquettes zéodratées, ainsi que l évaluation du risque qu elles représentent pour le receveur seront à fournir afin de déboucher sur un produit transfusionnelPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF