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Changes in microtubule polymerization and dynamics due to the tubulin alterations are factors that can affect the sensitivity of tumors to microtubule-disrupting agents. 2-methoxyestradiol (2ME2), an antiproliferative and antiangiogenic agent, depolymerizes microtubules at above 10 μM. At low concentrations, 2ME2 leads to G2/M cell cycle arrest followed by apoptosis, which is believed to be due to altered microtubule dynamics. Using a MDA-MB-435 cancer cell line resistant to 2-methoxyestradiol (2ME2R), we have previously shown that 2ME2R cells expressing two acquired β-tubulin mutations failed to depolymerize microtubules upon exposure of higher concentrations of 2ME2. In this study, we investigated whether the presence of these mutations can also contribute to altered cell cycle response in 2ME2R cells. Using flow cytometry analysis, we first showed that 2ME2R cells treated at 1 μM for 16 hours were insensitive to 2ME2-induced G2/M cell cycle arrest and to apoptosis. In contrast, treatment of parental cells with 2ME2 led to an accumulation of cells at the G2/M phase and resulted in apoptosis. Further evaluation of cyclin B1 and p34cdc2, two G2/M regulatory proteins that control the entrance to and exit from M-phase, revealed 2ME2-dependent upregulation of cyclin B1, activation of p34cdc2 kinase, and Bcl-2 phosphorylation in parental cells but not 2ME2R cells. Indirect immunofluorescence microscopy also confirmed accumulation of 2ME2-treated parental cells but not 2ME2R cells in prophase and metaphase with nuclear cyclin B1. To further determine whether the inability of 2ME2 to activate cyclin B1/p34cdc2 in 2ME2R cells correlates with altered tubulin status, extracts containing microtubule-associated proteins were prepared from both parental and 2ME2R cells in the presence or absence of 2ME2. Immunoblotting analysis demonstrated that treatment with 2ME2 increased cyclin B1: microtubule association in parental cells but not in 2ME2R cells. Taken together, these results suggest that the presence of tubulin mutations in 2ME2R cells affects the association of cyclin B1/p34cdc2 complex with the microtubules resulting in altered cell cycle response in 2ME2R cells.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]