J BIOCHEM MOLECULAR TOXICOLOGY Volume 19, Number 5, 2005 Activation of Caspase-3-Dependent and -Independent Pathways During 7-Ketocholesterol- and 7-Hydroxycholesterol-Induced Cell Death: A Morphological and Biochemical Study C´ eline Prunet, 1 St´ ephanie Lemaire-Ewing, 1 Franck M´ en´ etrier, 2 Dominique N´ eel, 1 and G´ erard Lizard 1 1 Inserm U498/IFR 100, CHU/H ˆ opital du Bocage, Laboratoire de Biochimie M´ edicale, BP 77908, 21079 Dijon Cedex, France; E-mail: Gerard.Lizard@chu-dijon.fr 2 IFR 100 Inserm, Centre de Microscopie Appliqu´ e` a la Biologie et ` a la M´ edecine, 7 Bd Jeanne d’Arc, 21033 Dijon Cedex, France Received 5 May 2005; revised 21 June 2005; accepted 22 June 2005 ABSTRACT: On treatment with 7-ketocholesterol (7-keto) or 7-hydroxycholesterol (7-OH), which are major oxysterols in atherosclerotic plaques, the si- multaneous identification of oncotic and apoptotic cells suggests that these compounds activate differ- ent metabolic pathways leading to various modes of cell death. With U937, MCF-7 (caspase-3 deficient), and MCF-7/c3 cells (stably transfected with caspase-3), we demonstrate that caspase-3 is essential for caspase- 9, -7, and -8 activation, for Bid degradation medi- ating mitochondrial cytochrome c release, for cleav- age of poly(ADP-ribose) polymerase and inhibitor of the caspase-activated deoxyribonuclease, and, at least in part, for internucleosomal DNA fragmen- tation. The crucial role of caspase-3 was supported by the use of z-VAD-fmk and z-DEVD-fmk, which abolished apoptosis and the associated events. How- ever, inactivation or lack of caspase-3 did not inhibit 7-keto- and 7-OH-induced cell death characterized by staining with propidium iodide, and loss of mi- tochondrial potential. The mitochondrial release of apoptosis-inducing factor and endonuclease G was in- dependent of the caspase-3 status, which conversely played major roles in the morphological aspects of dead cells. We conclude that caspase-3 is essential to trigger 7-keto- and 7-OH-induced apoptosis, and that these oxysterols simultaneously activate caspase- 3-dependent and/or -independent modes of cell death. C  2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:311–326, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI10.1002/jbt.20096 KEYWORDS: Apoptosis; AIF; Bid; Caspases; Cell Death; Endo G; ICAD; PARP; Oxysterols Correspondence to: G´ erard Lizard. c  2005 Wiley Periodicals, Inc. INTRODUCTION Oxidized low-density lipoproteins (Ox-LDL) and some of their lipid constituents (oxysterols, oxidized fatty acids, aldehydes, and lysophospholipids) [1] have numerous detrimental effects against the cells of the vascular wall (endothelial cells, smooth muscle cells, macrophages) including the induction of radical oxy- gen species overproduction [2], cytokine secretion [3], and cell death [4]. Oxysterols such as 7-ketocholesterol (7-keto) and 7-hydroxycholesterol (7-OH) (present at enhanced levels in atherosclerotic plaques [5] and plasma of patients with higher atherogenic risk or with coronary artery disease) [6,7] are known to mediate the various effects of Ox-LDL [4] (when taken in isolation or used in mixture [5,8]). These compounds are capable of inducing a complex mode of cell death, combined with mechanisms of apoptosis [9,10]. Therefore, it has been hypothesized that atheromatous lesions might repre- sent “death zones,” containing toxic materials such as oxysterols, in which monocytes/macrophages would become dysfunctional and apoptotic [11]. Nowadays, apoptosis is described as being mediated at least by three major pathways: the death receptor (or extrin- sic pathway), the mitochondrial (or intrinsic pathway), and the endoplasmic reticulum signaling pathways [12], all of which required the activation of caspases [13] according to chronology, depending on the inducer considered [14]. Moreover, despite a better under- standing of cell death mechanisms, confusion between the several cell demises, including the recently de- fined caspase-independent cell death [15], contributes to obscure the field of oxysterol-induced cell dam- ages. Therefore, as apoptosis of vascular cells occur- ring in atherosclerotic plaques is believed to promote 311