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Title: Involvement of mitochondria and caspase-3 in ET-18-OCH3-induced apoptosis of human leukemic cells
Authors: Gajate, Consuelo, Santos-Beneit, Antonio M., Lazaro, Maria del Carmen, Hernández de Rojas, Alma, Mollinedo, Faustino
Issue Date: 2000
Publisher: Wiley-Blackwell
Abstract: The induction of cell death in leukemic HL-60 cells by the ether lipid I-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3; edelfosine) followed the typical apoptotic changes in ultrastructural morphology, including blebbing, chromatin condensation, nuclear membrane breakdown and extensive vacuolation. Using a cytofluorimetric approach, we found that ET-18-OCH3 induced disruption of the mitochondrial transmembrane potential (ΔΨ(m)) followed by production of reactive oxygen species (ROS) and DNA fragmentation in leukemic cells. ET-18-OCH3 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-ribose) polymerase (PARP). ET-18-OCH3 analogues unable to induce apoptosis failed to disrupt ΔΨ(m) and to activate caspase-3. ET-18-OCH3-resistant Jurkat cells generated from sensitive Jurkat cells showed no caspase-3 activation and did not undergo ΔΨ(m) disruption upon ET-18-OCH3 incubation. Cyclosporin A partially inhibited ΔΨ(m) dissipation, caspase activation and apoptosis in ET-18-OCH3-treated leukemic cells. Overexpression of bcl-2 by gene transfer prevented ΔΨ(m) collapse, ROS generation, caspase activation and apoptosis in ET-18-OCH3-treated leukemic T cells. Pretreatment with the caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone prevented ET-18-OCH3- induced PARP proteolysis and DNA fragmentation, but not ΔΨ(m) dissipation. ET-18-OCH3 did not affect the expression of caspases and bcl-2-related genes. ET-18-OCH3-induced apoptosis did not require protein synthesis. Our data indicate that ΔΨ(m) dissipation and caspase-3 activation are critical events of the apoptotic cascade triggered by the antitumor ether lipid ET.18.OCH3, and that the sequence of events in the apoptotic action of ET- 18-OCH3 on human leukemic cells is: ΔΨ(m) disruption, caspase-3 activation and internucleosomal DNA degradation.
Publisher version (URL): http://dx.doi.org/10.1002/(SICI)1097-0215(20000415)86:2<208::AID-IJC10>3.0.CO;2-E
URI: http://hdl.handle.net/10261/60787
Identifiers: issn: 0020-7136
e-issn: 1097-0215
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Citation: International Journal of Cancer 86(2): 208-218 (2000)
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