Human mutations that confer paclitaxel resistance

Abstract

The involvement of tubulin mutations as a cause of clinical drug resistance has been intensely debated in recent years. In the studies described here, we used transfection to test whether beta1-tubulin mutations and polymorphisms found in cancer patients are able to confer resistance to drugs that target microtubules. Three of four mutations (A185T, A248V, R306C, but not G437S) that we tested caused paclitaxel resistance, as indicated by the following observations: (a) essentially 100% of cells selected in paclitaxel contained transfected mutant tubulin; (b) paclitaxel resistance could be turned off using tetracycline to turn off transgene expression; (c) paclitaxel resistance increased as mutant tubulin production increased. All the paclitaxel resistance mutations disrupted microtubule assembly, conferred increased sensitivity to microtubule-disruptive drugs, and produced defects in mitosis. The results are consistent with a mechanism in which tubulin mutations alter microtubule stability in a way that counteracts drug action. These studies show that human tumor cells can acquire spontaneous mutations in beta1-tubulin that cause resistance to paclitaxel, and suggest that patients with some polymorphisms in beta1-tubulin may require higher drug concentrations for effective therapy.

Figure 1

Survival of transfected cells in paclitaxel. Cells stably expressing wild-type (WT) or mutant HAβ1-tubulin were incubated in 200 nM paclitaxel (Ptx) (left two wells), with or without tetracycline (Tet), and grown until visible colonies appeared. Controls containing 50 times fewer cells and incubated in tetracycline alone are shown in the rightmost wells to estimate the relative number of cells seeded. Colonies were stained with methylene blue and photographed.

Figure 2

Production of mutant HAβ1-tubulin in paclitaxel resistant cells. G418 resistant cells (lanes 1, 4, and 7) were grown overnight without tetracycline. Cells selected for resistance to 200 nM (lanes 2, 5, and 8) or 300 nM (lanes 3, 6, and 9) paclitaxel were maintained in selection medium. The cells were lysed in SDS and the proteins were separated on an SDS gel, transferred onto nitrocellulose, and probed with antibodies to β-tubulin and actin. Ratios of the exogenous HAβ1-tubulin to the endogenous β-tubulin (HAβ1/β) for each lane are shown at the bottom of the figure.

Figure 3

Immunofluorescence microscopy of cells transfected with wild-type and mutant HAβ1-tubulin. Cells transfected with wild-type (WT, panel A) or the four mutant HAβ1-tubulin cDNAs [A185T (B), A248V (C), R306C (D), and G437S (E)] were stained with an antibody to the HA tag and with DAPI, a fluorescent molecule that binds to DNA. Note that the 3 mutant tubulins that confer paclitaxel resistance (A185T, A248V, and R306C) reduce microtubule density and cause problems with chromosome segregation as indicated by the abnormal nuclear morphologies (Fig. 3 B-D). Bar = 10 μm.

Figure 4

Microtubule content in transfected cell lines. Stable cell lines expressing wild-type or mutant HAβ1-tubulin cDNA were tested for their relative microtubule content. Cell lines transfected with wild-type or G437S HAβ1-tubulin were selected in G418 and screened for clones with high expression of the exogenous tubulin. Cell lines with the other mutations were selected in 200 or 300 nM paclitaxel but tested in the absence of the drug. The data were calculated from three independent experiments with triplicate samples, and represent the percentage of total tubulin found in the microtubule fraction. Error bars indicate the standard deviation.

Figure 5

Sensitivity of transfected cells to drugs that target microtubules. An equal number of cells (100-300) that stably express wild-type or mutant HAβ1-tubulin cDNA were seeded into replicate wells in a 24-well dish with increasing concentrations of paclitaxel (A), epothilone A (B), colcemid (C), or vinblastine (D). The cells were grown until visible colonies appeared (7 days), and were then stained with methylene blue. To quantify the data, dye was extracted with SDS and the absorbance was read at 630 nm. The relative growth was calculated by dividing the absorbance at a given drug concentration by the absorbance in the absence of drug, and the quotient was plotted against the drug concentration. Each data point represents the average of 3 independent experiments. The IC50s and standard deviations are summarized in Table 1. Filled circles and solid line, wild-type; open circles and short-dash line, A185T; open squares and dash-dot line, A248V; open triangles and long-dash line, R306C; filled squares and dotted line, G437S.

Figure 6

Tubulin structure. The structure was drawn with MacPymol(50) using the published atomic coordinates 1JFF (31). Magenta, β subunit; blue, α subunit; gray spheres, paclitaxel; pink spheres, GDP/GTP, green, mutant residues. G437S could not be shown because it is located in the carboxy terminal tail for which no structure is available. (A) lateral view; (B) axial view with indicated orientation relative to the inside and outside of a microtubule.