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. 2011 May;2(5):368-77.
doi: 10.18632/oncotarget.250.

Class III β-tubulin counteracts the ability of paclitaxel to inhibit cell migration

Anutosh Ganguly  1 , Hailing Yang, Fernando Cabral
Affiliations

Class III β-tubulin counteracts the ability of paclitaxel to inhibit cell migration

Anutosh Ganguly et al. Oncotarget. 2011 May.

Abstract

Class III β-tubulin (β3) is associated with tumor aggressiveness, resistance to therapy, and patient relapse. To elucidate its action, we tested β3's effect on cell migration. Expression of β3 in HeLa and MCF-7 did not alter the intrinsic rate of cell migration, but it prevented the inhibition of migration by low, nontoxic concentrations of paclitaxel. The effects on cell motility were confirmed in CHO cells with tetracycline regulated expression of β3. Cell migration and microtubule dynamics were inhibited by similar concentrations of paclitaxel, but required a 5-10 fold higher drug concentration when β3 was expressed. The directionality of migration was normal in paclitaxel, but cells spent more time in a "paused" state during which there was no net movement. These studies support a model in which paclitaxel inhibits cell migration by suppressing microtubule dynamics and β3-tubulin counteracts paclitaxel action by maintaining microtubule dynamic activity. The results provide a potential explanation for the aggressiveness of β3-expressing tumors.

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Figures

Figure 1
Figure 1. β3-Tubulin production in human cancer cell lines
(A) CHO (lane 1), MCF7 (lane 2), HeLa (lane 3), and DU145 (lane 4) cells were lysed and analyzed on western blots with antibodies to β3-tubulin and actin. (B) The indicated cell lines were stained with antibodies specific for α-tubulin and β3-tubulin. Note the heterogeneity of the cells with respect to β3-tubulin staining. Arrows for DU145 indicate the rare presence of cells that are β3 negative. Bar, 50 μm.
Figure 2
Figure 2. Paclitaxel inhibition of cell migration
(A) A mixed culture of wild-type CHO cells and HAβ3-5 cells was scratched to make a wound and the cells were allowed to migrate for 24 h in the absence or presence of 10 nM paclitaxel. The cells were then stained for α-tubulin (red), HAβ3-tubulin (green), and DNA (blue). Arrows indicate the direction of movement. Bar, 50 μm. (B) Cells at the edge of the wound were scored for the presence or absence of HAβ3 expression and the percentage of HAβ3 containing cells was calculated from 20 random fields each containing approximately 20 cells along the leading edge. The experiment was repeated 3 times. (C) In a separate experiment, a pure culture was used to measure the rate of HAβ3-5 cell migration into a scratch wound with and without 10 nM paclitaxel in the presence (open bar) and absence (solid bar) of tetracycline. (D) A transwell assay was used to measure the migration of cells from the upper to the lower chamber over a 6 h period at various paclitaxel concentrations. The graph was generated by defining the number of cells that migrated to the lower chamber at 0 nM paclitaxel as 100% and expressing the data for the other concentrations relative to the zero control. Error bars, SD. *p < 0.05 relative to the WT control was considered significant.
Figure 3
Figure 3. Paclitaxel suppression of microtubule dynamics
Wild-type and HAβ3-5 cells were transfected with EGFP-MAP4 and microtubules were imaged every 5 s in the presence of the indicated concentrations of paclitaxel (Ptx). Microtubule lengths from an arbitrary internal reference point were plotted against time to describe the growth and shortening of the microtubule plus end. Each line represents a separate microtubule. Note that the position of the line on the y-axis is arbitrary and does not represent the actual total length of the microtubule.
Figure 4
Figure 4. Effect of β3-tubulin expression on microtubule dynamics and cell migration
Wild-type CHO cells (WT, circles) and HAβ3-5 cells (squares) were treated with varying concentrations of paclitaxel and the effects of the drug on microtubule dynamicity (solid symbols) and cell migration (open symbols) were measured. Note the relative resistance of both microtubule dynamics and cell migration to the inhibitory effects of paclitaxel in HAβ3-5 cells.
Figure 5
Figure 5. Paclitaxel effects on cell movement
Wild-type and HAβ3-5 cells migrating into a wound in the presence and absence of paclitaxel were tracked for 5 h by marking the X and Y positions of the nucleus relative to a fixed internal reference point at 15 min intervals (open circles). For cases when the cell was paused (no movement) during one 15 min interval, the position is marked with a closed circle. When there was no movement during two 15 min intervals, the position is marked with an asterisk. Note that directionality didn't change, but the frequency of pauses greatly increased at inhibitory concentrations of paclitaxel.
Figure 6
Figure 6. Calculation of cell movement parameters
Microtubule tracks similar to those in Figure 5 were used to measure cell velocity (using only intervals during which the cells actually moved, solid bars), cell migration (1/2 the rate at which the wound closed, open bars), and frequency of pauses (number of intervals/h in which there was no cell movement, shaded bars).
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References

    1. Luduena RF. Multiple forms of tubulin: different gene products and covalent modifications. Internatl Rev Cytol. 1998;178:207–75. - PubMed
    1. Sullivan KF. Structure and utilization of tubulin isotypes. Ann Rev Cell Biol. 1988;4:687–716. - PubMed
    1. Fulton C, Simpson PA. Selective synthesis and utilization of flagellar tubulin. The multi-tubulin hypothesis. In: Goldman R, Pollard T, Rosenbaum J, editors. Cell Motility. New York: Cold Spring Harbor Press; 1976. pp. 987–1006.
    1. Bond JF, Fridovich-Keil JL, Pillus L, Mulligan RC, Solomon F. A chicken-yeast chimeric β-tubulin protein is incorporated into mouse microtubules in vivo. Cell. 1986;44:461–8. - PubMed
    1. Joshi HC, Yen TJ, Cleveland DW. In vivo coassembly of a divergent β-tubulin subunit (cβ6) into microtubules of different function. J Cell Biol. 1987;105:2179–90. - PMC - PubMed

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