Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 9 (11), 2914-23

Paclitaxel-dependent Cell Lines Reveal a Novel Drug Activity

Affiliations

Paclitaxel-dependent Cell Lines Reveal a Novel Drug Activity

Anutosh Ganguly et al. Mol Cancer Ther.

Abstract

We previously described the isolation of Tax 18 and Tax 11-6, two paclitaxel-dependent cell lines that assemble low amounts of microtubule polymer and require the drug for cell division. In the present studies, fluorescence time-lapse microscopy was used to measure microtubule dynamic instability behavior in these cells. The mutations were found to cause small decreases in microtubule growth and shortening, but the changes seemed unable to explain the defects in microtubule polymer levels or cell division. Moreover, paclitaxel further suppressed microtubule dynamics at low drug concentrations that were insufficient to rescue the mutant phenotype. Wild-type (WT) cells treated with similar low drug concentrations also had highly suppressed microtubules, yet experienced no problems with cell division. Thus, the effects of paclitaxel on microtubule dynamics seemed to be unrelated to cell division in both WT and mutant cell lines. The higher drug concentrations needed to rescue the mutant phenotype instead inhibited the formation of unstable microtubule fragments that appeared at high frequency in the drug-dependent, but not WT, cell lines. Live cell imaging revealed that the fragments were generated by microtubule detachment from centrosomes, a process that was reversed by paclitaxel. We conclude that paclitaxel rescues mutant cell division by inhibiting the detachment of microtubule minus ends from centrosomes rather than by altering plus-end microtubule dynamics.

Figures

Figure 1
Figure 1
Effect of paclitaxel on microtubule organization and nuclear morphology. Wild-type and paclitaxel dependent mutants Tax 18 and Tax 11-6 were treated for 2 d with 0-100 nM paclitaxel (Ptx) and viewed by immunofluorescence using an antibody to α-tubulin. Arrowheads point to some of the abundant microtubule fragments in the mutant cell lines cultured in low concentrations of paclitaxel. Scale bar equals 10 μm. Insets, nuclear morphology. DNA was stained with DAPI and the image sizes were reduced 50% relative to their corresponding cells.
Figure 2
Figure 2
Microtubule life history plots. Wild-type (WT) and mutant CHO cell lines were transfected with EGFP-MAP4 and fluorescent microtubules were imaged at 5 s intervals in the absence (A-C) or presence (D-F) of paclitaxel. A concentration of paclitaxel that is minimally toxic (50 nM) was used to treat the wild-type cells; whereas mutant cells were treated with the minimum drug concentrations (50 nM for Tax 11-6 and 100 nM for Tax 18) needed to rescue cell division. The graphs show changes in the position of randomly chosen microtubule plus-ends during the period of observation. Each line represents a single microtubule. Note that the y-axis represents the distance of the plus-end from an arbitrary reference point and does not represent the actual total length of the microtubule. In some cases, plots were arbitrarily distributed along the y-axis to avoid extensive overlap.
Figure 3
Figure 3
Effect of paclitaxel on cell division, microtubule fragmentation, and microtubule dynamics. Wild-type cells (A), Tax 18 (B), and Tax 11-6 (C) were incubated with increasing concentrations of paclitaxel for 2 d. Each culture was then tested for the percentage of cells that were multinucleated (a measure of cells that were unable to complete cell division; filled circles), the percentage of cells with fragmented microtubules (open circles), and dynamicity (a measure of dynamic behavior; filled squares). Values for a variety of parameters that describe the dynamic behavior can be found in Supplementary Tables S2-S4.
Figure 4
Figure 4
Microtubule detachment from centrosomes. (A) Paclitaxel dependent mutants Tax 11-6 and Tax 18 were grown for 2 d without paclitaxel, transfected with EGFP-MAP4 and viewed by live cell imaging. Fluorescent images taken 5 s apart were deconvolved to improve contrast. Note detachment and shortening from the minus end of a long microtubule in Tax 11-6 (arrows, upper panels); and detachment and translocation of a newly nucleated microtubule in Tax 18 (arrows, lower panels). Asterisks mark the centrosomal area of the cells. Scale bar equals 5 μm. (B) Cells treated with the indicated concentrations of paclitaxel for 2 d were viewed by time-lapse fluorescence microscopy. Microtubules that detached from the centrosome were counted from 25-40 min of time-lapse video recordings representing 5 cells in 2 separate experiments, and the rate was calculated as the number of detachments/cell/min. Error bars indicate the standard deviation from the mean.
Figure 5
Figure 5
Microtubule nucleation. (A) Wild-type (WT) and mutant cells (Tax 18 and Tax 11-6) were grown 2 d in the absence of paclitaxel, transfected with EB1-GFP, and viewed at 5 s intervals. Comets representing the plus-ends of newly nucleated microtubules in the area around the centrosome are shown. Scale bar equals 2 μm. (B) Comets newly appearing within a 30 μm2 circular area around the centrosome were counted and averaged from 25 successive images in each of 3-5 separate cells. The y-axis represents the average number of comets per frame. Error bars represent the standard deviation from the mean.
Figure 6
Figure 6
Model to explain drug resistance and dependence. PtxR, paclitaxel resistant mutant; PtxD, paclitaxel dependent mutant; CmdR, colcemid resistant mutant; CmdD, colcemid dependent mutant; [Cmd] or [Ptx], drug concentration. Dashed vertical lines, boundaries between normal proliferation and toxicity (defects in mitosis and cell division).

Similar articles

See all similar articles

Cited by 35 PubMed Central articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback