Original Contribution Differential effects of clusterin/apolipoprotein J on cellular growth and survival Ioannis P. Trougakos a , Magda Lourda a , Georgia Agiostratidou a , Dimitris Kletsas b , Efstathios S. Gonos a, * a Laboratory of Molecular & Cellular Aging, Institute of Biological Research & Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., Athens 11635, Greece b Laboratory of Cell Proliferation & Aging, Institute of Biology, National Centre for Scientific Research, bDemokritosQ, Athens 15310, Greece Received 3 June 2004; revised 10 August 2004; accepted 28 October 2004 Available online 24 November 2004 Abstract The secreted clusterin/apolipoprotein J (CLU) protein form is a ubiquitously expressed heterodimeric glycoprotein which is differentially regulated in many severe physiological disturbance states including cell death, ageing, cancer progression, and various neurological diseases. Despite extensive efforts CLU function remains an enigma, the main cause being the intriguingly distinct and usually opposed functions in various cell types and tissues. In the current report we investigated the effects of CLU on cellular growth and survival in three human osteosarcoma (OS) cell lines, namely KH OS, Sa OS, and U-2 OS that express very low, moderate, and high endogenous steady-state CLU amounts, respectively. We found that exposure of these established OS cell lines or primary OS cells to genotoxic stress results in CLU gene induction at distinct levels that correlate negatively to CLU endogenous amounts. Following CLU-forced overexpression by means of an artificial transgene, we found that although extracellular CLU inhibits cell death in all three OS cell lines, intracellular CLU has different effects on cellular proliferation and survival in these cell lines. Transgenic KH OS cell lines adapted to moderate intracellular CLU levels were growth-retarded and became resistant to genotoxic and oxidative stress. In contrast, transgenic Sa OS and U2 OS cell lines adapted to high intracellular CLU amounts were sensitive to genotoxic and oxidative stress. In these two cell lines, the proapoptotic CLU function could be rescued by caspase inhibition. To monitor the immediate effects of heterologous CLU overexpression prior to cell adaptation, we performed transient transfections in all three OS cell lines. We found that induction of high intracellular CLU amounts increases spontaneous apoptosis in KH OS cells and reduces DNA synthesis in all three cell lines assayed. On the basis of these novel findings we propose that although extracellular CLU as well as intracellular CLU at low/moderate levels is cytoprotective, CLU may become highly cytostatic and/or cytotoxic if it accumulates intracellularly in high amounts either by direct synthesis or by uptake from the extracellular milieu. D 2004 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Clusterin/apolipoprotein J; Genotoxic stress; Necrosis; Oxidative stress; Free radicals Introduction Human CLU 1 is a heterodimeric glycoprotein that was initially purified from serum [1] and identified as an apolipoprotein [2]. Following these studies CLU has been found in all human body fluids analyzed [3]. Not only does CLU function as an apolipoprotein but it is also implicated in additional intra- or extracellular processes. The CLU gene encodes for a 449 amino acid polypeptide where the first 22 amino acids represent the classical hydrophobic secretory signal sequence. Maturation of the primary translation molecule includes disulfide bonding and subsequent con- version to a high-mannose endoplasmic reticulum-associ- ated form of ~60-kDa; extensive additional N-linked glycozylation and proteolytical cleavage in the trans-Golgi 0891-5849/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2004.10.038 Abbreviations: ANOVA, analysis of variance; CLU, clusterin/apolipo- protein J; DMEM, Dulbecco’s modified Eagle’s medium; DXR, doxor- ubicin (adriamycin); HDOs, human diploid osteoblasts; HSPs, heat shock proteins; OS, osteosarcoma; PBS, phosphate-buffered saline; PDs, pop- ulation doublings; SD, standard deviation. * Corresponding author. Fax: +30 210 7273677. E-mail address: sgonos@eie.gr (E.S. Gonos). Free Radical Biology & Medicine 38 (2005) 436 – 449 www.elsevier.com/locate/freeradbiomed