FEBS 29471 FEBS Letters 579 (2005) 2411–2415 Apoptosis repressor with caspase recruitment domain (ARC) is expressed in cancer cells and localizes to nuclei Mi Wanga, Suparna Qanungoa, Michael T. Crowb, Michiko Watanabec, Anna-Liisa Nieminena,* a Department of Anatomy and Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA b Division of Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, Johns Hopkins University, Baltimore, MD 21224, USA c Department of Pediatrics, Rainbow Babies and ChildrenÕs Hospital, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA Received 14 February 2005; accepted 2 March 2005 Available online 29 March 2005 Edited by Veli-Pekka Lehto and Bax regulate inhibition of apoptosis in a CARD depen- Abstract Apoptosis repressor with caspase recruitment domain is expressed at high levels in brain and myogenic tissues, consis- dent manner [2,6,7]. Therefore, ARC inhibits both extrinsic tent with a role to inhibit apoptosis in the terminally differenti- and intrinsic cell death pathways. Basal levels of ARC in ated cells. Expression of ARC in cancers is not known. In this cardiomyocytes, neurons, and skeletal muscle fibers serve to study, we reported that ARC was highly expressed in various repress apoptosis in these terminally differentiated cells. non-myogenic and non-neurogenic human and rat cancer cell Knockdown of endogenous ARC facilitates death-inducing lines. Unexpectedly, ARC was localized almost exclusively to signaling complex assembly and triggers spontaneous Bax acti- the nuclei of cancer cells, which was unlike the cytoplasmic local- vation and apoptosis [6]. Expression of ARC in cancer cells ization of ARC in non-cancer cells. Furthermore, nuclear ARC has not been reported. Here, we show that ARC is expressed in cancer cells did not co-localize with nucleolus protein of in various human and rat cancer cells. However, in contrast 30 kDa, an alternatively spliced ARC isoform. These findings to the non-cancer cells, ARC is localized almost exclusively indicate that ARC is distributed differently in cancer cells than non-cancer cells and thus might play a role in neoplastic trans- to nuclei. formation. Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 2. Materials and methods Keywords: Apoptosis repressor with caspase recruitment domain; Apoptosis; Cancer cells 2.1. Cell culture Human MIA PaCa-2 pancreatic carcinoma, U-87 glioblastoma, HeLa cervical carcinoma, MDA-MB-231 mammary gland adenocarci- noma, DU 145 prostate carcinoma, BJAB and DG-75 lymphoma cells; non-transformed H9c2 embryonic rat heart derived cells, rat N1-S1, McA-RH7777, and ASD30 hepatoma, and PC6 pheochromocytoma cells were grown in DulbeccoÕs modified EagleÕs medium (DMEM). HCT 116 wild type human colorectal carcinoma cells as well as +/À À/À 1. Introduction HCT 116 Bax and HCT Bax cells were maintained in McCoyÕs 5a medium. SH-SY5Y human neuroblastoma cells were maintained in DMEM/F-12 medium. All media were purchased from Gibco and Apoptosis plays a critical role in adult tissue homeostasis. supplemented with 10% fetal bovine serum, 50 units/ml of penicillin The failure of cells to undergo apoptotic cell death is a factor and 50 lg/ml of streptomycin. Primary cultured rat hippocampal neu- in the pathogenesis of various human diseases, including can- rons were maintained in Neurobasal-A containing 2% B27 supplement, cers, autoimmune diseases, and viral infections [1]. The human 2 mM Glutamax-1. Cells were incubated in a humidified 37 °C incuba- tor containing 5% CO and 95% air. apoptosis repressor with caspase recruitment domain (ARC) 2 gene was first identified by screening for proteins with homol- ogy to the caspase recruitment domain (CARD) of caspase-9 2.2. Western blot analysis [2]. ARC is highly expressed in brain and myogenic tissues, Cell and tissue extracts were prepared in ice-cold RIPA lysis buffer (150 mM NaCl, 1 mM EGTA, 1% sodium deoxycholate, 1% Triton e.g., skeletal muscle and heart [2,3]. Endogenous ARC is pre- X-100, and 50 mM Tris–Cl, pH 8.0) supplemented with a cocktail dominantly cytoplasmic in rat embryonic heart H9c2 cells of protease inhibitors (Roche Diagnostics). Fresh human skeletal and mouse skeletal muscle tissue [3,4]. ARC protects cells muscle was obtained from the Human Tissue Procurement Depart- against apoptosis triggered by several stimuli, including hydro- ment, University Hospitals of Cleveland. Protein samples (50 lg) were diluted in sample buffer (200 mM Tris–Cl, 15% glycerol, 10% gen peroxide, hypoxia, and Fas ligand [3,5,6]. Protein–protein SDS, 5% b-mercaptoethanol, and 0.01% phenol blue, pH 6.8) and re- interactions of ARC with Fas, Fas-associated death domain solved on 12% SDS–PAGE gel. Proteins were then transferred and immobilized onto PVDF membranes and probed with a polyclonal anti-ARC antibody and anti-rabbit secondary antibody. ARC anti- *Corresponding author. Fax: +1 216 368 8919. body was raised against a GST-ARC fusion protein and was affinity E-mail address: [email protected] (A.-L. Nieminen). purified using the C-terminal proline–glutamic acid (P/E)-rich domain [8]. b-Tubulin was detected with a monoclonal anti-b-tubulin anti- Abbreviations: ARC, apoptosis repressor with CARD domain; CARD, body (Sigma, 1:1000) and anti-mouse secondary antibody. YY1 caspase recruitment domain; CK2, casein kinase II; Nop30, nucleolus and NQO1 were detected with monoclonal anti-YY1 antibody (Santa protein of 30 kDa; NQO-1, NAD(P)H:quinone oxidoreductase 1; Cruz, 1:1000) and anti-NQO1 antibody (Santa Cruz, 1:2000), NLS, nuclear localization signal; YY-1, YinYang 1 respectively. 0014-5793/$30.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.03.040 2412 M. Wang et al. / FEBS Letters 579 (2005) 2411–2415 2.3. Nuclear and cytoplasmic extracts 2.6. Statistical analysis The nuclear and cytoplasmic extracts were prepared as previously Data are presented as means ± S.E.M. from three independent described with minor modifications [9]. Briefly, cells cultured on 150- experiments. Differences were assessed by two-tailed StudentÕs t test mm Petri dishes were scraped into 1 ml of ice-cold PBS. Cells were cen- with Instat Software (GraphPAD, San Diego, CA). A P value < 0.05 trifuged at 4000 · g for 5 min at 4 °C, and cell pellets were lysed in was considered to be statistically significant. 300 ll of buffer (20 mM HEPES, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.1% Triton X-100, pH 7.6) on ice. After 20 min incubation, supernatants were collected as cytoplas- mic extracts after centrifugation (4000 · g for 5 min at 4 °C). Pellets 3. Results and discussion were lysed in 30 ll of buffer (20 mM HEPES, 20% glycerol, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.1% Triton X-100, pH 7.6). After 20 min incubation on ice, supernatants were collected as nu- ARC is highly expressed in heart, skeletal muscle and brain clear extracts after centrifugation (16000 · g for 15 min at 4 °C). but is deficient in liver, lung, kidney, pancreas, placenta, and various lymphoid-hematopoietic tissues [2,5]. In order to 2.4. Immunocytochemistry examine whether ARC is expressed in human cancer cells, HCT 116, MDA-MB 231, and MIA PaCa-2 cells cultured on glass we assessed protein expression of ARC in a series of human coverslips were fixed with 1% paraformaldehyde overnight at 4 °C cancer cell lines. Equal amounts of protein from whole cell ly- and permeabilized in 0.05% Triton X-100 for 5 min at room tempera- sates prepared from 10 human cancer cell lines were loaded ture. After washes, cells were blocked with 1% BSA for 2 h, followed by incubation with primary antibody against ARC overnight at onto SDS–PAGE gels and analyzed by Western blot with an 4 °C. Cells were then washed with PBS followed by incubation with anti-ARC antibody (Fig. 1). Among these human cancer cell Alexa Fluor 488 or 543 anti-rabbit IgG for 1 h at room temperature. lines, ARC protein was detected in nine cell lines: MIA Hoechst 33342 (1 lg/ml) was included to stain nuclei. PaCa-2 pancreatic carcinoma (Fig. 1, lane 2); HCT 116 colo- Samples of human skeletal muscles were cut into 1 cm3 blocks and fixed overnight with 1% paraformaldehyde at 4 °C. Blocks were placed rectal carcinoma (lane 3); MDA 231 breast carcinoma (lane successively into 10%, 20% and 30% sucrose, respectively for 8 h at 4); A549 lung carcinoma (lane 5); HeLa cervical carcinoma 4 °C and frozen in embedding medium cooled on dry ice (Tissue- (lane 6); DU 145 prostate carcinoma (lane 7); U-87 glioblas- Tek). Frozen tissue sections were permeabilized with 0.05% Triton toma (lane 9); BJAB and DG-75 lymphoma (lane 10 and X-100 for 5 min and blocked with 10% goat serum and 1% BSA in 11). ARC was not detected in SH-SY5Y neuroblastoma (lane PBS for 2 h at room temperature. After incubation in primary anti- body against ARC for 2 h at room temperature, sections were washed 8). Tubulin was used as a loading control and was variable be- with PBS, followed by incubation with anti-rabbit Alexa Fluor 488 or tween different cell lines and tissue types. This likely reflects 543 for 1 h. Images of Alexa Fluor 488 fluorescence was collected using different levels of tubulin protein expression in the different cell 488-nm excitation and >505-nm emission filters. Alexa Fluor 543 fluo- types. For example, tubulin-rich brain tissue showed the dens- rescence was collected using 543-nm excitation and >560-nm emission filters.