7 research outputs found
Differential role of FL-BID and t-BID during verotoxin-1-induced apoptosis in Burkitt's lymphoma cells
Funding Information: Acknowledgements We thank Yann Lécluse (Imaging and Cytometry Platform, Institut Gustave Roussy) for expert technical assistance in performing flow cytometry analyses. We are very grateful to Hana Raslova and Julie RiviÚre (INSERM UMR 1170, Institut Gustave-Roussy) for their help in producing lentivirus and to Evelyne May and Martine Raphaël (UMR 8126 CNRS) for helpful discussions and suggestions. This work was supported by grants from the Fondation de France 2014 00047509 (JW), the Ligue National Contre le Cancer (doctoral fellowship to JD) and the Fondation ARC (doctoral fellowship to EH).Peer reviewe
Gb3/CD77 receptor : VT-1 apoptotic signaling pathway analysis in Burkitt lymphoma cells and research of endogens ligands
Le glycolipide Gb3/CD77 qui est fortement exprimĂ© en surface des cellules de lymphome de Burkitt (LB) est le rĂ©cepteur dâune toxine bactĂ©rienne la VĂ©rotoxine-1 (VT-1). Notre Ă©quipe a montrĂ© prĂ©cĂ©demment que, dans les cellules de LB, la VT-1 induit une cascade apoptotique mettant en jeu les caspases et la mitochondrie. Mon travail a consistĂ© Ă poursuivre lâanalyse des bases molĂ©culaires de ce processus, notamment en mâintĂ©ressant au rĂŽle de la protĂ©ine pro-apoptotique Bid. Bid est un membre de la famille Bcl-2 qui est clivĂ© par la caspase-8 au cours de lâapoptose et dont la forme tronquĂ©e (t-Bid) se relocalise Ă la mitochondrie. GrĂące Ă lâutilisation de clones cellulaires de LB oĂč Bid a Ă©tĂ© inhibĂ©e puis rĂ©exprimĂ©e sous forme non clivable et dâun inhibiteur de la caspase-8, nous avons montrĂ© que lors de lâapoptose induite par la VT1 : 1) la protĂ©ine entiĂšre Bid (full-length Bid ou FL-Bid) contrĂŽle lâactivation de protĂ©ines pro-apoptotiques Bax et de Bak ; 2) t-Bid et FL-Bid sont, toutes les deux, impliquĂ©es dans la libĂ©ration des protĂ©ines pro-apoptotiques (cytochrome c et Smac/DIABLO) de lâespace intermembranaire de la mitochondrie vers le cytosol ; 3) FL-Bid contrĂŽle lâhomodimĂ©risation de Bax et de Bak qui contribuerait Ă la libĂ©ration initiale du cytochrome c et de Smac/DIABLO alors que t-Bid est nĂ©cessaire Ă lâhĂ©tĂ©rodimĂ©risation de Bax et Bak qui permettrait lâamplification de cette libĂ©ration. Lâensemble de ces rĂ©sultats montre donc une coopĂ©ration fonctionnelle entre Bax et Bak au cours de lâapoptose induite par la VT-1 et surtout met en Ă©vidence que lâactivation de la voie caspase-8/t-Bid nâest absolument pas requise pour initier la mort cellulaire.Gb3/CD77 est aussi exprimĂ© Ă la surface de certains lymphocytes B normaux, oĂč il constitue un marqueur de diffĂ©renciation mais sa fonction endogĂšne reste encore indĂ©terminĂ©e. Une deuxiĂšme partie de mon travail a consistĂ© Ă essayer dâidentifier le ligand physiologique de Gb3/CD77 pour comprendre son rĂŽle biologique. GrĂące Ă une analyse en spectromĂ©trie de masse, nous avons identifiĂ© deux protĂ©ines potentiellement partenaires de Gb3/CD77 : la galectine-7 et la protĂ©ine S100A11.The Gb3/CD77 glycolipid, which is strongly expressed in Burkitt's lymphoma (BL) cells, is a receptor for the bacterial toxin Verotoxin-1 (VT-1). Previously, our group has shown that VT-1 induces an apoptotic pathway in BL cells which is dependent on caspases and mitochondria. Here, we provide new insights into this pathway. A pro-apoptotic member in the Bcl-2 family, Bid is cleaved by caspase-8 and its truncated form t-Bid is translocated to mitochondria. Using LB cell clones where Bid was inhibited prior to being reexpressed as a non-cleavable mutated form (BID D59A) and a caspase-8 inhibitor to explore VT-1-induced apoptosis, we showed that 1) the full length Bid (FL-Bid) controls the activation of pro-apoptotic proteins Bax and Bak; 2) Both t-Bid and FL-Bid are involved in the release of pro-apoptotic proteins (cytochrome c and Smac/DIABLO) from the mitochondrial intermembrane space to the cytosol; 3) FL-Bid controls the homo-oligomerization of both Bax and Bak, likely contributing to the initial release of cytochrome c and Smac/DIABLO while t-Bid is needed for their hetero-oligomerization followed by amplification of the release. Together, these results reveal a functional cooperation between Bax and Bak during VT-1-induced apoptosis and, most importantly, that activation of caspase-8 and t-Bid is not required to induce the onset of cell death. Gb3/CD77 is also expressed in a proportion of normal B-lymphocytes where it constitutes a differentiation marker but whose function remains uncharacterized. In an effort to look for physiological ligands, we have used a biochemical approach followed by mass spectrometry analysis. Two proteins have been identified as potentially Gb3/CD77 partners, namely galectin-7 and protein S100A11
Analyse des voies d'apoptose induites par la toxine bactérienne la vérotoxine-1 dans les cellules de lymphome de Burkitt
La vĂ©rotoxine-1 (VT-1) est une toxine de la famille des Shiga-toxines qui produite par diffĂ©rentes souches d E.coli, et qui a por rĂ©cepteur le glycolipide (Gb3). Gb3 est fortement exprimĂ© Ă la surface des cellules de lymphome de Burkitt (LB), une cascade apoptotique mettant en jeu les caspases et la mitochondrie. Afin de caractĂ©riser plus prĂ©cisĂ©ment les voies de signalisation impliquĂ©es dans cette apoptose, nous nous sommes intĂ©ressĂ©es aux rĂŽles de protĂ©ines pro-apoptotiques de la famille de Bcl-2 : Bid et Bax. Nous avons montrĂ©, dans notre modĂšle, que l activation de Bax (changement de conformation et relocalisation mitochondriale) est importante pour la relocalisation des protĂ©ines cytochrome c et Smac de l espace intermembranaire mitochondrial vers le cytosol. Cette activation, partiellement contrĂŽlĂ©e par Bid/tBid, est impliquĂ©e dans le dĂ©clenchement de l apoptose induite par VT-1. Bid/tBid sont aussi cruciaux pour la formation d hĂ©tĂ©rodimĂšres de Bax/Bak dont la fonction prĂ©cise reste Ă dĂ©terminer. Par ailleurs, nos rĂ©sultats montrent que la surexpression de Bcl-2 et l inhibition de Bid emĂȘchent le clivage de la caspase-8 et de Bid, ce qui suggĂšre que la caspase-8 est activĂ©e en amont et en aval (boucle d amplification) de la mitochondrie.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF
The BCLâ2 family protein inhibitor ABTâ737 as an additional tool for the treatment of EBVâassociated postâtransplant lymphoproliferative disorders
International audiencePost-transplant lymphoproliferative disorders (PTLD) and Burkitt's lymphoma (BL) are B-cell malignancies strongly associated with Epstein-Barr virus (EBV) infection. In these lymphoproliferative disorders, EBV infection induces an increase in the expression of the anti-apoptotic protein BCL-2. Given its chemoprotective effect, BCL-2 constitutes an attractive target for new therapeutic strategies for EBV-positive B-cell malignancies. Here, we show that ABT-737, a small inhibitor of BCL-2, BCL-X(L), and BCL-w, strongly induced apoptosis in vitro in EBV-positive lymphoblastoid cell lines (which is a model for PTLD), whereas BL was less sensitive. ABT-737 reduced tumor growth and increased the overall survival of mice in a xenograft model of PTLD but had no effect on BL xenograft mice. ABT-737 combined with a low dose of cyclophosphamide, a major component of the conventional CHOP chemotherapy regimen for BL patients, reduced tumor growth during treatment but failed to improve the overall survival of BL xenograft mice. By contrast, the combination of ABT-737 and rituximab, one of the main options for the treatment of PTLD, was highly efficient and induced approximately 70% remission in PTLD xenograft mice. These results suggest that the use of agents targeting BCL-2, either alone or in combination with other conventional drugs, represents a novel promising approach for post-transplant EBV-positive B lymphoproliferative disorders
Verotoxin-1-Induced ER Stress Triggers Apoptotic or Survival Pathways in Burkitt Lymphoma Cells
Shiga toxins (Stxs) expressed by the enterohaemorrhagic Escherichia coli and enteric Shigella dysenteriae 1 pathogens are protein synthesis inhibitors. Stxs have been shown to induce apoptosis via the activation of extrinsic and intrinsic pathways in many cell types (epithelial, endothelial, and B cells) but the link between the protein synthesis inhibition and caspase activation is still unclear. Endoplasmic reticulum (ER) stress induced by the inhibition of protein synthesis may be this missing link. Here, we show that the treatment of Burkitt lymphoma (BL) cells with verotoxin-1 (VT-1 or Stx1) consistently induced the ER stress response by activation of IRE1 and ATF6—two ER stress sensors—followed by increased expression of the transcription factor C/REB homologous protein (CHOP). However, our data suggest that, although ER stress is systematically induced by VT-1 in BL cells, its role in cell death appears to be cell specific and can be the opposite: ER stress may enhance VT-1-induced apoptosis through CHOP or play a protective role through ER-phagy, depending on the cell line. Several engineered Stxs are currently under investigation as potential anti-cancer agents. Our results suggest that a better understanding of the signaling pathways induced by Stxs is needed before using them in the clinic
Metabolic Activation of Prasugrel: Nature of the Two Competitive Pathways Resulting in the Opening of Its Thiophene Ring
The mechanism generally admitted for the bioactivation
of the antithrombotic
prodrug, prasugrel, <b>1c</b>, is its two-step enzymatic conversion
into a biologically active thiol metabolite. The first step is an
esterase-catalyzed hydrolysis of its acetate function leading to a
thiolactone metabolite <b>2c</b>. The second step was described
as a cytochrome P450 (P450)-dependent oxidative opening of the thiolactone
ring of <b>2c</b>, with intermediate formation of a reactive
sulfenic acid metabolite that is eventually reduced to the corresponding
active thiol <b>3c</b>. This article describes a detailed study
of the metabolism of <b>1c</b> by human liver microsomes and
human sera, with an analysis by HPLC-MS under conditions allowing
a complete separation of the thiol metabolite isomers, after derivatization
with 3âČ-methoxy phenacyl bromide. It shows that there are two
competing metabolic pathways for the opening of the <b>2c</b> thiolactone ring. The major one, which was previously described,
results from a P450- and NADPH-dependent redox bioactivation of <b>2c</b> and leads to <b>3c</b>, two previously reported thiol
diastereomers bearing an exocyclic double bond. It occurs with NADPH-supplemented
human liver microsomes but not with human sera. The second one results
from a hydrolysis of <b>2c</b> and leads to an isomer of <b>3c</b>, <b>3c endo</b>, in which the double bond has migrated
from an exocyclic to an endocyclic position in the piperidine ring.
It occurs both with human liver microsomes and human sera, and does
not require NADPH. However, it requires Ca<sup>2+</sup> and is inhibited
by paraoxon, which suggests that it is catalyzed by a thioesterase
such as PON-1. Chemical experiments have confirmed that hydrolytic
opening of thiolactone <b>2c</b> exclusively leads to derivatives
of the endo thiol isomer <b>3c endo</b>