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 arrest

We have found that the bicyclic colchicine analogue 2-methoxy-5-(2',3',4'-t rimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) induced a dose- and time -dependent apoptotic response in human leukemic cells. MTC and colchicine r apidly disrupted the microtubule integrity and arrested cells at the G(2)-M phase before the onset of apoptosis, These responses were mediated by micr otubule inhibition because 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)propiony l] one and lumicolchicine, inactive analogues of MTC and colchicine, respec tively, were unable to promote microtubule disassembly, cell cycle arrest, and apoptosis, Although 1 mu M R-ITC induced a complete microtubule disrupt ion after 1 h of incubation in human leukemic HL-60 cells that led to an ac cumulation of cells at the G(2)-M phase, MTC-induced apoptosis occurred aft er 9 h of treatment. This indicates the existence of a rather long lag betw een microtubule disruption and the onset of apoptosis. Unlike colchicine, t he removal of MTC during this lag resulted in rapid microtubule repolymeriz ation, followed by restoration of normal cell cycle and cell growth. MTC, b ut not 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl) propionyl]amino]-2,4,6-cycl oheptatrien-1-one induced c-jun expression as well as c-Jun NH2-terminal ki nase and caspase activation, indicating that these signaling pathways are t riggered by the specific action of MTC on microtubules, Caspase inhibition prevented MTC-induced apoptosis. Overexpression of bcl-2 or bcl-x(L) by gen e transfer in human erythroleukemic HEL cells abrogated MTC-induced apoptos is, but cells remained arrested in G(2)-M, suggesting that bcl-2 and bcl-x( L) block the signaling pathway between G(2)-M arrest and triggering of apop tosis, MTC-treated bcl-2 and bcl-x(L)-transfected HEL cells recovered their rapacity to proliferate after MTC removal. These results indicate that mic rotubule inhibition induces G(2)-M arrest and apoptosis in leukemic cells, showing a lag phase between G(2)-M arrest and the onset of apoptosis, regul ated by bcl-2 and bcl-x(L) during which MTC displays a reversible action on microtubule depolymerization and G(2)-M cell cycle arrest. Thus, MTC is a potent apoptotic inducer on human leukemic cells and shows a remarkable rev ersible action on microtubule network and cell cycle before commitment for apoptosis is reached.