INVOLVEMENT OF MITOCHONDRIA AND CASPASE-3 IN ET-18-OCH 3 -INDUCED APOPTOSIS OF HUMAN LEUKEMIC CELLS Consuelo GAJATE 1,2 , Antonio M. SANTOS-BENEIT 1,2 , Antonio MACHO 3 , Maria del Carmen LAZARO 2 , Alma HERNANDEZ-DE ROJAS 2 , Manuel MODOLELL 4 , Eduardo MU ˜ NOZ 3 and Faustino MOLLINEDO 1,2 * 1 Centro de Investigacio ´n del Ca ´ncer, Instituto de Biologı ´a Molecular y Celular del Ca ´ncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain 2 Instituto de Biologı ´a y Gene ´tica Molecular, Facultad de Medicina, CSIC-Universidad de Valladolid, Valladolid, Spain 3 Departamento de Fisiologı ´a e Inmunologı ´a, Facultad de Medicina, Universidad de Co ´rdoba, Co ´rdoba, Spain 4 Max-Planck-Institut fu ¨r Immunbiologie, Freiburg, Germany The induction of cell death in leukemic HL-60 cells by the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3- phosphocholine (ET-18-OCH 3 ; edelfosine) followed the typical apoptotic changes in ultrastructural morphology, including blebbing, chromatin condensation, nuclear mem- brane breakdown and extensive vacuolation. Using a cyt- ofluorimetric approach, we found that ET-18-OCH 3 induced disruption of the mitochondrial transmembrane potential ( m ) followed by production of reactive oxygen species (ROS) and DNA fragmentation in leukemic cells. ET-18- OCH 3 also induced caspase-3 activation in human leukemic cells, as assessed by cleavage of caspase-3 into the p17 active form and cleavage of the caspase-3 substrate poly(ADP-ri- bose) polymerase (PARP). ET-18-OCH 3 analogues unable to induce apoptosis failed to disrupt  m and to activate caspase-3. ET-18-OCH 3 -resistant Jurkat cells generated from sensitive Jurkat cells showed no caspase-3 activation and did not undergo  m disruption upon ET-18-OCH 3 incubation. Cyclosporin A partially inhibited  m dissipation, caspase activation and apoptosis in ET-18-OCH 3 -treated leukemic cells. Overexpression of bcl-2 by gene transfer prevented  m collapse, ROS generation, caspase activation and apo- ptosis in ET-18-OCH 3 -treated leukemic T cells. Pretreat- ment with the caspase inhibitor Z-Asp-2,6-dichlorobenzoy- loxymethylketone prevented ET-18-OCH 3 -induced PARP proteolysis and DNA fragmentation, but not  m dissipa- tion. ET-18-OCH 3 did not affect the expression of caspases and bcl-2-related genes. ET-18-OCH 3 -induced apoptosis did not require protein synthesis. Our data indicate that  m dissipation and caspase-3 activation are critical events of the apoptotic cascade triggered by the antitumor ether lipid ET-18-OCH 3 , and that the sequence of events in the apopto- tic action of ET-18-OCH 3 on human leukemic cells is:  m disruption, caspase-3 activation and internucleosomal DNA degradation. Int. J. Cancer 86:208 –218, 2000. © 2000 Wiley-Liss, Inc. Antitumor ether-linked lysophospholipid analogues are a novel class of promising cancer chemotherapeutic drugs that do not interact with DNA, and induce apoptosis in cancer cells (Munder and Westphal, 1990; Houlihan et al., 1995; Mollinedo et al., 1997). Some of these compounds are scheduled for, or currently undergoing, phase I/II clinical evaluation (Houlihan et al., 1995). The ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phospho- choline (ET-18-OCH 3 ; edelfosine) is a synthetic analogue of 2-ly- sophosphatidylcholine, exerts a selective cytotoxic action against transformed cells (Munder and Westphal, 1990; Houlihan et al., 1995; Mollinedo et al., 1997), and has become the effective standard and prototype of the antitumor ether phospholipids. En- couraging clinical research on the use of ET-18-OCH 3 in purging leukemic bone marrow prior to autologous bone marrow trans- plantation has been reported (Koenigsmann et al., 1996; Yamazaki and Sieber, 1997). ET-18-OCH 3 is a potent inducer of apoptosis in tumor cells (Mollinedo et al., 1993, 1997; Diomede et al., 1993), sparing normal cells (Mollinedo et al., 1997), and this apoptotic action appears to be the major antitumor effect of ET-18-OCH 3 . After programmed cell death is triggered, different members of 2 growing gene families, namely Bcl-2-related genes and caspases, mainly regulate the process of apoptosis. Following the initial identification of the bcl-2 gene at the chromosomal breakpoint of t(4;18)-bearing B-cell lymphoma, several homologous proteins have subsequently been identified which comprise the growing Bcl-2 protein family. Some members of this Bcl-2 protein family inhibit apoptosis (Bcl-2, Bcl-X L , Bcl-w, Bfl-1) and other promote apoptosis (Bax, Bak, Bik, Bad, Bcl-X S ). The so-called caspase family comprises a growing group of at least 14 cysteine proteases showing homology with the mammalian protein interleukin-1- converting enzyme (ICE) and exerting an effector function in programmed cell death. The name of caspase for these ICE-like proteases was selected to indicate 2 catalytic properties of these enzymes: the “c” denoting a cysteine protease, and the “aspase” referring to their ability to cleave after an aspartic residue. Involvement of mitochondria in apoptotic processes has been demonstrated following induction of cell death with a wide num- ber of stimuli (Kroemer et al., 1997). Early after induction of apoptosis, a loss of mitochondrial transmembrane potential ( m ) can be detected together with the formation of permeability tran- sition pores (Kroemer et al., 1997). This leads to the release of cytochrome c into the cytoplasm where it results in activation of caspase-3, presumably with the help of Apaf-1 following activa- tion of caspase-9 (Li et al., 1997). The mechanism by which ET-18-OCH 3 engages selectively the suicide apparatus remains to be elucidated, and little is known about the regulatory events that render cells susceptible to undergo programmed cell death upon ET-18-OCH 3 treatment. We have previously found evidence for the highly selective apoptotic effect of ET-18-OCH 3 for malignant cells (Mollinedo et al., 1997) and postulated that this selectivity is causally related to the cellular uptake of the compound (Mollinedo et al., 1997). We have also previously found that ectopic overexpression of bcl-2 or bcl-x L prevents apoptosis induced by ET-18-OCH 3 , but not its cellular uptake (Mollinedo et al., 1997), suggesting that high levels of Bcl-2 and Bcl-X L can block the intracellular signaling route lead- ing to cell death triggered by ET-18-OCH 3 . ET-18-OCH 3 arrests mitogenic signals, such as MAPK/ERK (Zhou et al., 1996), and induces apoptotic signals, such as persistent activation of c-Jun kinase (JNK) (Gajate et al., 1998), suggesting these actions can be involved in the antitumor mechanism of the ether lipid. On the Grant sponsor: INKEYSA and Ministerio de Industria y Energı ´a of Spain; Grant number: CDTI 97-0355; Grant sponsor: European Commis- sion and Comisi´ on Interminisiterial de Ciencia y Tecnologı ´a (CICYT); Grant number 1FD97-0622; Grant sponsor: Junta de Castilla y Le ´ on; Grant number: VA32/99; Grant sponsor: CICYT; Grant number: SAF98/0047; Grant sponsor: Direcci´ on General de Investigaci´ on Cientı ´fica y T´ ecnica (DGICYT); Grant number: PB95-0713; Grant sponsor: Fondo de Investi- gaci´ on Sanitaria (FIS96/1434). *Correspondence to: Instituto de Biologı ´a y Gen´ etica Molecular, Fac- ultad de Medicina, CSIC-Universidad de Valladolid, C/ Ram´ on y Cajal, 7, E-47005 Valladolid, Spain. Fax: +34-983-423588. E-mail: fmollin@med.uva.es Received 3 September 1999; Revised 28 October 1999. Int. J. Cancer: 86, 208 –218 (2000) © 2000 Wiley-Liss, Inc. Publication of the International Union Against Cancer