| 10738248 |
Gajate C, Santos-Beneit AM, Macho A, Lazaro M, Hernandez-De Rojas A, Modolell M, Munoz E, Mollinedo F: Involvement of mitochondria and caspase-3 in ET-18-OCH (3)-induced apoptosis of human leukemic cells. Int J Cancer. 2000 Apr 15;86(2):208-18.
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 membrane breakdown and extensive vacuolation. Using a cytofluorimetric approach, we found that ET-18-OCH (3) induced disruption of the mitochondrial transmembrane potential (DeltaPsi (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-ribose) polymerase (PARP). ET-18-OCH (3) analogues unable to induce apoptosis failed to disrupt DeltaPsi (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 DeltaPsi (m) disruption upon ET-18-OCH (3) incubation. Cyclosporin A partially inhibited DeltaPsi (m) dissipation, caspase activation and apoptosis in ET-18-OCH (3)-treated leukemic cells. Overexpression of bcl-2 by gene transfer prevented DeltaPsi (m) collapse, ROS generation, caspase activation and apoptosis in ET-18-OCH (3)-treated leukemic T cells. Pretreatment with the caspase inhibitor Z-Asp-2, 6-dichlorobenzoyloxymethylketone prevented ET-18-OCH (3)-induced PARP proteolysis and DNA fragmentation, but not DeltaPsi (m) dissipation. 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 DeltaPsi (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 apoptotic action of ET-18-OCH (3) on human leukemic cells is: DeltaPsi (m) disruption, caspase-3 activation and internucleosomal DNA degradation. |
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