Dependency of 2-methoxyestradiol-induced mitochondrial apoptosis on mitotic spindle network impairment and prometaphase arrest in human Jurkat T cells.
The present study sought to determine the correlation between 2-methoxyestradiol (2-MeO-E2)-induced cell cycle arrest and 2-MeO-E2-induced apoptosis. Exposure of Jurkat T cell clone (JT/Neo) to 2-MeO-E2 (0.5-1.0 μM) caused G2/M arrest, Bak activation, Δψm loss, caspase-9 and -3 activation, PARP cleavage, intracellular ROS accumulation, and apoptotic DNA fragmentation, whereas none of these events except for G2/M arrest were induced in Jurkat T cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, Cdk1 phosphorylation at Thr-161 and dephosphorylation at Tyr-15, up-regulation of cyclin B1 expression, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation at Ser-159/Thr-163, and Bim phosphorylation were detected irrespective of Bcl-2 overexpression. Concomitant treatment of JT/Neo cells with 2-MeO-E2 and the G1/S blocking agent aphidicolin resulted in G1/S arrest and abrogation of all apoptotic events, including Cdk1 activation, phosphorylation of Bcl-2, Mcl-1 and Bim, and ROS accumulation. The 2-MeO-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor, but not by an Aurora A kinase (AURKA), Aurora B kinase (AURKB), JNK, or p38 MAPK inhibitor. Immunofluorescence microscopic analysis revealed that 2-MeO-E2-induced mitotic arrest was caused by mitotic spindle network impairment and prometaphase arrest. Whereas 10-20 μM 2-MeO-E2 reduced the proportion of intracellular polymeric tubulin to monomeric tubulin, 0.5-5.0 μM 2-MeO-E2 increased it. These results demonstrate that the apoptogenic effect of 2-MeO-E2 (0.5-1.0 μM) was attributable to mitotic spindle defect-mediated prometaphase arrest, Cdk1 activation, phosphorylation of Bcl-2, Mcl-1, and Bim, and activation of Bak and mitochondria-dependent caspase cascade.
Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea.
Seung Tae
ST
Lee
Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea.
Ji Young
JY
Lee
Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea.
Cho Rong
CR
Han
Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea; Daegu Science High School, Daegu 706-852, Republic of Korea.
Yoon Hee
YH
Kim
Institute of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea.
Do Youn
do Y
Jun
Center for Translational Studies, Medical Services, Veterans Administration Medical Center, Department of Veteran Affairs, Washington, DC 20422, USA.
Dennis
D
Taub
Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea. Electronic address: ykim@knu.ac.kr.
Young Ho
YH
Kim
D000077584:2-Methoxyestradiol; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D004789:Enzyme Activation; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects; D006801:Humans; D019169:Jurkat Cells; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000502:physiology; D008938:Mitosis; D010766:Phosphorylation; D049468:Prometaphase / Q000187:drug effects*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000187:drug effects* / Q000502:physiology
D000970:Antineoplastic Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D004958:Estradiol; D000077584:2-Methoxyestradiol; D053148:Caspase 3
Cdk1 activation; Mitochondrial apoptosis; Mitotic spindle damage; Phosphorylation of Bcl-2 family proteins; Prometaphase arrest