Resistance to Taxol in lung cancer cells associated with increased microtubule dynamics

Abstract

Microtubule dynamics are crucial for mitotic spindle assembly and chromosome movement. Suppression of dynamics by Taxol appears responsible for the drug's potent ability to inhibit mitosis and cell proliferation. Although Taxol is an important chemotherapeutic agent, development of resistance limits its efficacy. To examine the role of microtubule dynamics in Taxol resistance, we measured the dynamic instability of individual rhodamine-labeled microtubules in Taxol-sensitive and -resistant living human cancer cells. Taxol-resistant A549-T12 and -T24 cell lines were selected from a human lung carcinoma cell line, A549. They are, respectively, 9- and 17-fold resistant to Taxol and require low concentrations of Taxol for proliferation. We found that microtubule dynamic instability was significantly increased in the Taxol-resistant cells. For example, with A549-T12 cells in the absence of added Taxol, microtubule dynamicity increased 57% as compared with A549 cells. The length and rate of shortening excursions increased 75 and 59%, respectively. These parameters were further increased in A549-T24 cells, with overall dynamicity increasing by 167% compared with parental cells. Thus, the decreased Taxol-sensitivity of these cells can be explained by their increased microtubule dynamics. When grown without Taxol, A549-T12 cells were blocked at the metaphase/anaphase transition and displayed abnormal mitotic spindles with uncongressed chromosomes. In the presence of 2-12 nM Taxol, the cells grew normally, suggesting that mitotic block resulted from excessive microtubule dynamics. These results indicate that microtubule dynamics play an important role in Taxol resistance, and that both excessively rapid dynamics and suppressed dynamics impair mitotic spindle function and inhibit proliferation.

Figure 1

Dynamic behavior of microtubules in living A549 cells. Arrows indicate three microtubules that undergo shortening events. Time is indicated as min:s.

Figure 2

Life history plots (length changes over a duration of ≈2.5 min) of individual dynamic microtubules in (A) A549 cells and (B) A549-T12 cells in the absence of added Taxol.

Figure 3

Mitotic spindle organization. (A and D) A549 cells; (B and E) A549-T12 cells in 12 nM Taxol; (C and F) A549-T12 cells in the absence of added Taxol; (G and H) anaphase in A549-T12 cells in 12 nM Taxol. (A–C, G) Spindle microtubules as shown by antitubulin immunofluorescence. (D–F, H) Chromosomes of the same cells as shown by 4,6-diamino-2-phenylindole stain. (Bar = 10 μm.)

Figure 4

Microtubule organization in interphase cells. (A) A549 cells; (B) A549-T12 in 12 nM Taxol; (C) A549-T12 in the absence of added Taxol. Antitubulin immunofluorescence. Arrows in B point to Taxol-induced microtubule bundles. (Bar = 10 μm.)