In situ localization of paclitaxel binding structures: Labeling with a paclitaxel fluorescent analogue.
Microtubules are a major component of cell cytoskeleton. Microtubules constitute the cellular target of paclitaxel. The interaction of paclitaxel with microtubules causes an increase in tubulin polymerization and microtubules stabilization, leading to a G2/M phase cell cycle arrest and cell death by apoptosis. Three paclitaxel fluorescent analogues were prepared by introducing fluorescein or BODIPY moiety onto the 2' of the 7-carbon. These were then used to study the interaction of paclitaxel with its cellular binding site and the microtubule network was visualized directly by fluorescence microscopy. Free paclitaxel was able to inhibit 7-FITC paclitaxel binding, demonstrating that both products bind to the same site and possess similar biological properties. Using the carbon 7 derivatives, microtubules were labeled as cytoplasmic fibers extending centripedly. A few other cellular components such as nuclear membrane, nucleoli and some other cytoplasmic structures were also labeled. The labeling intensity was reduced by preincubation with free paclitaxel. The interaction of paclitaxel with microtubules was also investigated using flow cytometry. No binding of the 2'C derivative was detected, confirming that a free 2'C is a pre-requisite for paclitaxel microtubules interaction.
Bicamumpaka
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Department of Medical Biology, Faculty of Medicine, Laval University, Sainte Foy, Quebec G1K 7P4, Canada.
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