Antimitotic agent alters MIP levels In breast cancer cells

Abstract

Progression through mitosis requires a balance of active microtubule-interacting proteins (MIPs) that stabilize and destabilize microtubules in mitotic spindles. Molecular interactions between MIPs and tubulin or microtubules are important for cell cycle progression. Insights into these interactions can contribute to understanding the mechanisms underlying cell cycle interference by antimitotic agents that halt cell cycle progression in mitosis. We examined the direct interaction of the antimitotic agent, vinblastine with tubulin and stathmin using AUC, in order to understand how changes in stathmin levels during the cell cycle might affect the cellular drug response. Vinblastine acts during G2/M phase of the cell cycle and reduces microtubule dynamics. At high doses it destabilizes microtubules in mitotic spindles. We found in vitro that stathmin reduces the potency of vinblastine. Vinblastine was found to compete for tubulin-stathmin oligomers, at the same time as it induced tubulin spiral formation. To extend these data to a cellular context, we investigated changes in intracelluar MIP levels in response to paclitaxel, an antimitotic agent known to stabilize microtubules at high concentrations. Using qRT-PCR we found that paclitaxel treatment of human breast cancer MCF7 cells leads to a significant reduction in MAP4 and stathmin mRNA levels. Interestingly, the levels return to pre-paclitaxel treatment levels after a 4-day drug washout, suggesting that paclitaxel alters transcript levels. We found that the ratios of MAP4/stathmin increased after or during drug treatment. These data suggest that changes in MIPs levels alter the cellular response to drugs. These results also suggest that disruption of the cell cycle by antimitotic agents can alter the relative amounts of MIPs and thus affect the balance needed for normal progression through the cell cycle. These results must be taken into account when modeling the cellular response to antimitotic drugs.