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Induction of Apoptosis in Leukemic Cells by the Reversible Microtubule-disrupting Agent 2-Methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one: Protection by Bcl-2 and Bcl-X_L and Cell Cycle Arrest¹

Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Cientificas-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain [C. G., F. M.]; Instituto de Biología y Genética Molecular, Facultad de Medicina, Consejo Superior de Investigaciones Cientificas-Universidad de Valladolid, E-47005 Valladolid, Spain [C. G., F. M.]; and Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, E-28006 Madrid, Spain [I. B., J. M. A.]

We have found that the bicyclic colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) induced a dose- and time-dependent apoptotic response in human leukemic cells. MTC and colchicine rapidly disrupted the microtubule integrity and arrested cells at the G₂-M phase before the onset of apoptosis. These responses were mediated by microtubule inhibition because 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)propionyl]amino]-2,4,6-cycloheptatrien-1-one and lumicolchicine, inactive analogues of MTC and colchicine, respectively, were unable to promote microtubule disassembly, cell cycle arrest, and apoptosis. Although 1 µM MTC induced a complete microtubule disruption after 1 h of incubation in human leukemic HL-60 cells that led to an accumulation of cells at the G₂-M phase, MTC-induced apoptosis occurred after 9 h of treatment. This indicates the existence of a rather long lag between microtubule disruption and the onset of apoptosis. Unlike colchicine, the removal of MTC during this lag resulted in rapid microtubule repolymerization, followed by restoration of normal cell cycle and cell growth. MTC, but not 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)propionyl]amino]-2,4,6-cycloheptatrien-1-one, induced c-jun expression as well as c-Jun NH₂-terminal kinase and caspase activation, indicating that these signaling pathways are triggered by the specific action of MTC on microtubules. Caspase inhibition prevented MTC-induced apoptosis. Overexpression of bcl-2 or bcl-x_L by gene transfer in human erythroleukemic HEL cells abrogated MTC-induced apoptosis, but cells remained arrested in G₂-M, suggesting that bcl-2 and bcl-x_L block the signaling pathway between G₂-M arrest and triggering of apoptosis. MTC-treated bcl-2 and bcl-x_L-transfected HEL cells recovered their capacity to proliferate after MTC removal. These results indicate that microtubule inhibition induces G₂-M arrest and apoptosis in leukemic cells, showing a lag phase between G₂-M arrest and the onset of apoptosis, regulated by bcl-2 and bcl-x_L, during which MTC displays a reversible action on microtubule depolymerization and G₂-M cell cycle arrest. Thus, MTC is a potent apoptotic inducer on human leukemic cells and shows a remarkable reversible action on microtubule network and cell cycle before commitment for apoptosis is reached.

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