Triacsin C inhibits the formation of 1 H NMR-visible mobile lipids and lipid bodies in HuT 78 apoptotic cells Egidio Iorio a , Massimo Di Vito a , Francesca Spadaro a , Carlo Ramoni a , Emanuela Lococo b , Roberto Carnevale b , Luisa Lenti b , Roberto Strom c , Franca Podo a, * a Laboratory of Cell Biology, Istituto Superiore di Sanita `, Viale Regina Elena 299, 00161 Rome, Italy b Department of Experimental Medicine and Pathology, University of Rome ‘‘La Sapienza’’, 00185 Rome, Italy c Department of Cellular Biotechnology and Haematology, University of Rome ‘‘La Sapienza’’, 00161 Rome, Italy Received 7 February 2003; received in revised form 9 June 2003; accepted 24 July 2003 Abstract Nuclear magnetic resonance-visible mobile lipids (ML) have been reported to accumulate during cell apoptosis in vitro and in vivo. The biogenesis, biochemical nature and structure of these lipids are still under debate. In this study, a human lymphoblastoid cell line, HuT 78, was induced to apoptosis by exposure to anti-Fas monoclonal antibodies (a-Fas mAb) followed by incubation for different time intervals (1 – 24 h, hypodiploid cell fraction, H, varying from 1% to over 60%) either in the presence or in the absence of 5.0 AM Triacsin C (TRC), specific inhibitor of long-chain acyl-CoA synthetase (ACS). The increase of ML in apoptotic cells correlated linearly with H and was associated with: (a) accumulation of intracellular lipid bodies, detected by confocal laser scanning microscopy in lipophilic dye-stained cells; (b) increases, detected by thin-layer chromatography in total lipid extracts, in the relative abundance of triacylglycerides (TAG) and cholesteryl esters (CE), with corresponding decreases of phospholipids (PL). TRC completely abolished both ML and lipid body formation in anti-Fas-treated apoptotic cells, with concomitant reversion of TAG, CE and PL to control levels, but did not alter cell viability nor did it inhibit apoptosis. ML signals detected during anti-Fas-induced apoptosis therefore appear to originate from neutral lipids assembled in intracellular lipid bodies, synthesised from cellular acyl-CoA pools. D 2003 Elsevier B.V. All rights reserved. Keywords: Apoptosis; Lymphoblast; Mobile lipid; NMR; Triacsin C; Fas 1. Introduction Apoptosis is a tightly regulated form of physiological cell death in multicellular organisms, dependent upon the ex- pression of some cell-intrinsic, genetically controlled sui- cide machinery [1,2]. This cell death programme not only plays fundamental physiological roles in embryogenesis, tissue homeostasis, immune response and aging, but is also reputed to be involved in a number of pathological process- es including neurodegenerative disorders, autoimmune dis- eases, ischemia, oncogenesis and tumour response to therapy [3–9]. Although a definite pattern of all the molecular events leading to apoptosis is not yet available, an increasing level of knowledge is accumulating on this phenomenon, at the biological and biochemical level. 1 H NMR spectroscopy has recently added interesting novel information on the bio- chemistry of programmed cell death, by allowing non- invasive monitoring of alterations occurring in the energetic state, as well as in phospholipid and glucose metabolism [10–17] and by detecting the formation and accumulation of mobile lipids (ML) in intact apoptotic cells [18–22]. The production of these lipids, endowed of a sufficiently high rotational tumbling motion and segmental flexibility of their fatty acyl chains to be detected in the high-resolution nuclear magnetic resonance (NMR) time window, is not a peculiar feature of apoptotic cells, since the characteristic 1388-1981/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2003.07.001 Abbreviations: a-Fas mAb, anti-Fas monoclonal antibodies; ACS, acyl- CoA synthetase; CE, cholesteryl esters; CHOL, free cholesterol; CLSM, confocal laser scanning microscopy; CoA, coenzyme A; DAG, diacylgly- cerides; FFA, free fatty acids; HPTLC, high-performance thin-layer chromatography; ML, NMR-detectable mobile lipids; NMR, nuclear magnetic resonance; PC, phosphatidylcholine; PCho, phosphocholine; PEtn, phosphoethanolamine; PrI, propidium iodide; PL, phospholipids; TAG, triacylglycerides; TRC, Triacsin C * Corresponding author. Tel.: +39-6-4990-2686; fax: +39-6-4938- 7144. E-mail address: fpodo@iss.it (F. Podo). www.bba-direct.com Biochimica et Biophysica Acta 1634 (2003) 1 – 14