Phosphoinositide phosphatase SHIP-1 regulates apoptosis induced by edelfosine, Fas ligation and DNA damage in mouse lymphoma cells Maaike C. Alderliesten, Jeffrey B. Klarenbeek, Arnold H. van der Luit, Menno van Lummel, David R Jones, Shuraila Zerp, Nullin Divecha, Marcel Verheij, Wim J van Blitterswijk To cite this version: Maaike C. Alderliesten, Jeffrey B. Klarenbeek, Arnold H. van der Luit, Menno van Lummel, David R Jones, et al.. Phosphoinositide phosphatase SHIP-1 regulates apoptosis induced by edelfosine, Fas ligation and DNA damage in mouse lymphoma cells. Biochemical Journal, Portland Press, 2011, 440 (1), pp.127-135. 10.1042/BJ20110125. hal-00642836 HAL Id: hal-00642836 https://hal.archives-ouvertes.fr/hal-00642836 Submitted on 19 Nov 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Biochemical Journal Immediate Publication. Published on 27 Jul 2011 as manuscript BJ20110125 Phosphoinositide phosphatase SHIP-1 regulates apoptosis induced by edelfosine, Fas ligation and DNA damage in mouse lymphoma cells Maaike C. ALDERLIESTEN*, Jeffrey B. KLARENBEEK*, Arnold H. VAN DER ‡ LUIT*2, Menno VAN LUMMEL*3, David R. JONES , Shuraila ZERP*†, Nullin ‡ DIVECHA , Marcel VERHEIJ† and Wim J. VAN BLITTERSWIJK*1 *Division of Cell Biology (B5) and †Department of Radiotherapy, The Netherlands Cancer Institute/ Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX Amsterdam, The ‡ Netherlands, Paterson Institute for Cancer Research, Inositide laboratory, The University of Manchester, Wilmslow Road, M20 4BX Manchester, UK. Short title: SHIP-1 regulates apoptosis sensitivity Abbreviations used: ALP, alkyl-lysophospholipid; Akt/PKB, protein kinase B; FasL, Fas ligand; ERK, extracellular-signal-regulated kinase; PI3K, phosphoinositide 3-kinase; SHIP, SH2 domain-containing inositol 5-phosphatase; SM, sphingomyelin; SMS, sphingomyelin synthase. 1 To whom correspondence should be addressed (Tel: +31-756282239; Fax: +31-20- 5121944; E-mail: [email protected]) 2 Present address: Oncodesign, 20, rue Jean Mazen, BP 27627, 21076 Dijon Cedex, France 3 Present address: Department of Immunehematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20110125 Accepted Manuscript Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited Biochemical Journal Immediate Publication. Published on 27 Jul 2011 as manuscript BJ20110125 2 SYNOPSIS S49 mouse lymphoma cells undergo apoptosis in response to the alkyl- lysophospholipid (ALP) edelfosine, Fas/CD95 ligand (FasL) and DNA damage. Cells made resistant to ALP (S49AR) are defective in sphingomyelin synthesis and ALP uptake, and also have acquired resistance to FasL and DNA damage. However, these cells can be re-sensitized following prolonged culturing in the absence of ALP. The resistant cells show sustained ERK/Akt activity, consistent with enhanced survival signalling. In search of a common mediator of the observed cross- AR resistance, we found that S49 cells lacked the PtdIns(3,4,5)P3 phosphatase SHIP-1, a known regulator of the Akt survival pathway. Re-sensitization of the S49AR cells restored SHIP-1 expression as well as phosphoinositide and sphingomyelin levels. Knockdown of SHIP-1 mimicked the S49AR phenotype in terms of apoptosis cross- resistance, sphingomyelin deficiency and altered phosphoinositide levels. Collectively, our results suggest that SHIP-1 collaborates with sphingomyelin synthase to regulate lymphoma cell death irrespective of the nature of the apoptotic stimulus. Keywords: SHIP, alkyl-lysophospholipid; apoptosis resistance; Fas/CD95; DNA damage THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20110125 Accepted Manuscript Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited Biochemical Journal Immediate Publication. Published on 27 Jul 2011 as manuscript BJ20110125 3 INTRODUCTION Synthetic alkylphospholipids such as edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero- 3-phosphocholine; alkyl-lysophospholipid; ALP) are promising new anticancer agents that act on lipid-dependent signal transducing enzymes in tumor cell membranes rather than the DNA [1-4]. They easily insert in the lipid bilayer, accumulate in lipid rafts and persist due to poor degradation [5-7]. Interference with lipid metabolism and signal transduction leads to apoptosis, selectively in tumor cells [1,3,8]. The therapeutic value of these compounds appeared most prominent in combination with conventional DNA damaging regimens, such as -radiation [1,3,9-11]. We previously reported that, in S49 mouse lymphoma cells, ALP is internalized by raft-dependent endocytosis and inhibits the biosynthesis of major phospholipids, which induces apoptosis [6,7]. A variant cell line, S49AR, made resistant to ALP, showed impaired uptake of ALP and related alkylphospholipids [6,7]. As a result, they are not only resistant to apoptosis by these lipid compounds but, strikingly, also by ligation of the CD95/Fas death-receptor (FasL) [12]. Intriguingly, S49AR cells were deficient in the major raft lipid, sphingomyelin (SM), due to complete down-regulation of SM synthase-1 (SMS1) [13]. Although siRNA-induced down-regulation of SMS1 in S49 cells mimicked apoptosis resistance, reconstitution of SMS1 in S49AR cells failed to restore apoptosis sensitivity, which led to the conclusion that SMS1 deficiency is important but not sufficient to explain the apoptosis resistance to ALP and FasL [12]. The present paper reports on a second determinant of resistance and on the observation that S49AR cells are also cross-resistant to DNA damage. Resistance to apoptosis is often accompanied by up-regulation of survival signalling via ERK and protein kinase B (PKB)/Akt [1]. This notion was also confirmed here in our resistant S49AR cells. Using pharmacological inhibitors, we checked if activity of these kinases was essential for the apoptosis resistance, but found that this was only the case for resistance to DNA damage, not to ALP or FasL. Since Akt is thus not the common and sole determinant of cross-resistance to the various apoptotic stimuli, we looked upstream of this protein kinase and focused on phosphoinositides. SH2-containing inositol 5-phosphatase 1 (SHIP-1) is an important regulator of survival signalling [14-16]. It is mainly expressed in hematopoietic cells where it converts PtdIns(3,4,5)P3 to PtdIns(3,4)P2 and is as such a negative regulator of the PI3K/Akt signalling pathway [15]. Next to the catalytic (phosphatase) domain, SHIP-1 contains a number of structural motifs that allow physical interaction with other signalling proteins, such as an SH2 domain, proline-rich motifs that can bind SH3-containing proteins, and NPXY sites that, when tyrosine-phosphorylated, binds proteins containing a PTB domain, for example Shc [14,15]. THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20110125 Here we report that SHIP-1 is down-regulated in resistant S49AR cells and that, accordingly, PtdInsP3 level is increased, consistent with increased phosphorylation and thus activation of Akt and ERK1/2. Spontaneous re-sensitization of S49AR cells (yielding S49ARS cells) leads to regained expression of SHIP-1 and normalized phosphoinositide levels, comparable to S49 cells. Knocking-down SHIP-1 by siRNA mimics apoptosis resistance to DNA damage and FasL and partly the resistance to ALP. These data indicateAccepted that absence of SHIP-1 negatively Manuscriptregulates apoptosis induced by multiple stimuli leading to multi-stress-resistant lymphoma cells. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited Biochemical Journal Immediate Publication. Published on 27 Jul 2011 as manuscript BJ20110125 4 EXPERIMENTAL Materials ALP (edelfosine; Et-18-OCH3;1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) was purchased from BioMol (Plymouth Meeting, PA). [3H]edelfosine (3H-ALP; 58 Ci/mmol), was synthesized by Moravek Biochemicals (Brea, CA). [3H]1-sphingosine was synthesized by Piet Weber (DSM, Delft, The Netherlands). Etoposide was from Sigma. Tween-20 and Silica 60 TLC plates were from Merck (Darmstadt, Germany). Anti-Fas monoclonal antibody 7C10 was from CAMPRO Scientific BV (Veenendaal, The Netherlands). Protein G sepharose fast flow beads were from Amersham Biosciences (Roosendaal, The Netherlands); Soluble recombinant human Fas ligand (FasL; APO-1L) was from Alexis (Leiden, The Netherlands). Rabbit anti--actin, anti-pSer473-Akt, anti- pThr308-Akt, anti-Akt, anti-MAPK, anti-PTEN, LY294002, rapamycin and U0126 were from Cell Signaling Technology. AKTi1/2 and z-VAD-FMK (carbobenzoxy-valyl- alanyl-aspartyl-[O-methyl]-fluoromethylketone) were from Calbiochem (Darmstadt, Germany). Anti-Fas mAb Jo2 (hamster IgG2) was from BD Biosciences