Article Figures & Data
Figures
- Fig. 1.
Two-dimensional gel electrophoresis patterns of Colcemid- and vinblastine-resistant cells. Autoradiograms of the gels are oriented with the basic side to the left and the acidic side to the right. A, wild-type two-dimensional gel pattern shared by strains CV 8-1 and CV 2-3. v, vimentin; a, actin; α, α-tubulin; β, β-tubulin. Note that wild-type α-tubulin migrates as two spots with the same pI but different apparent molecular weights and that β-tubulin migrates as a single spot. B, a more basic β-tubulin spot (arrow) is seen in Cmd4, CV 1-1, and CV 1-9. C, β-tubulin with a lower apparent molecular weight (arrow) is seen in CV 7-1. D, β-tubulin with a more acidic pI (arrow) is seen in CV 4-4. E, two α-tubulin spots with a more basic pI (arrow) are seen in CV 2-8. F, two α-tubulin spots with a more acidic pI (arrow) are seen in CV 1-2, CV 1-5, CV 1-7, and VV 3-2. The identity of tubulin spots in these gels has been established by immunoreactivity with specific antibodies, peptide maps, and comigration with purified tubulin (13, 23, 24).
- Fig. 2.
Dose response of mutant CHO cells to Colcemid and vinblastine. Equal numbers of mutant or wild-type CHO cells (∼100–200) were plated in each of 6 wells in α-MEM containing an increasing concentration of drugs as labeled. After 7 days of growth, the medium was removed, and the cells were stained with 0.25% methylene blue.
- Fig. 3.
Dose response of drug-resistant cells to paclitaxel. Equal numbers of mutant or wild-type CHO cells (∼100–200) were plated in each of 6 wells in α-MEM containing an increasing concentration of paclitaxel as labeled. After 7 days of growth, the medium was removed, and the cells were stained with 0.25% methylene blue.
- Fig. 4.
Tubulin assembly in drug-resistant cells. Cells were lysed in microtubule-stabilizing buffer, soluble tubulin was separated from microtubules by centrifugation, and proteins in each fraction were separated by SDS-PAGE and transferred onto nitrocellulose. Tubulin in pellet (P) and supernatant (S) fractions was labeled with anti-tubulin antibodies followed by a Cy5-conjugated secondary antibody. The bands were detected by fluorescence using a STORM imager and quantified using Scion Image software. The amount of tubulin in the pellet was expressed as a percentage of total tubulin (S + P). The data shown represent averages from at least three independent experiments.
- Fig. 5.
Microtubule organization in drug-resistant CHO cells. Colcemid-resistant mutant CV 7-1 (A), wild-type CHO (B), or paclitaxel-resistant Tax 18 (C) or cells transfected with HA-tagged β1 tubulin cDNA containing a C211F mutation (D and E) or a D45Y mutation (F) were grown on glass coverslips. Soluble tubulin was pre-extracted using microtubule-stabilizing buffer, cells were fixed in methanol, and microtubules were stained with anti-α-tubulin antibodies (A−C) or antibodies to a HA epitope tag (D−F). Note that CV 7-1 (A) has a high density of microtubules forming bundles near the nucleus and extending to the cell periphery (arrow). Bar in C is 10 μm.
- Fig. 6.
Colcemid resistance of transfected cells. Approximately 50–100 stably transfected cells expressing wild-type (HAβwt) or mutant [HAβ(D45Y)] tubulin were seeded into replicate wells of a 24-well dish containing the indicated concentrations of Colcemid. After 7 days of growth, the medium was removed, and the cells were stained with 0.25% methylene blue. Note that the assay was carried out in the presence (+) or absence (−) of 1 μg/ml tetracycline to respectively inhibit or allow expression of the transgene.
- Fig. 7.
Sites of amino acid substitutions that stabilize micro- tubule assembly. A, ribbon diagram of an αβ-tubulin heterodimer with paclitaxel and GDP-bound β-subunit (red) and GTP-bound α-subunit (blue). Space-filling models of GDP and GTP are represented in gray, and paclitaxel is in lavender. The positions of altered residues are depicted as stick models in yellow (α-subunit) and green (β-subunit) and labeled with the amino acid substitution. B, atomic surface of a tubulin heterodimer illustrating the degree of exposure of altered residues to bulk solvent. Orientation of the molecule, coloring, and labels are as described for A. These representations show that in α-tubulin, Glu55 and His283 are located on surface loops, whereas Ala383 and Arg390 (in helix 11) are buried. In the β-subunit Asp45, Asp224, and Ser234 are partially solvent-exposed; Cys211 is buried near a lateral interface; and Lys350 is buried at the longitudinal intradimer interface. Residues Cys211, Asp224, and Ser234 are located within 8 Å of paclitaxel. Structural coordinates corresponding to the Protein Data Bank file 1TUB (42) were used to generate the model, and the illustration was prepared using InsightII software.
Tables
- Table 1
Properties of colcemid and vinblastine resistant cell lines
Cell line Two-dimensional gel change (new spot) Drug sensitivity Tubulin assembly (% of total tubulin) Paclitaxel Colcemid Wild-type No change Sa S 39 Tax 5-6 α1 Basic shift R SS 25 (P <0.01)b Cmd4 β1 Basic shift SSc Rc 50c CV 1-1 β1 Basic shift SS R 54 (P < 0.01) CV 7-1 β1 Lower molecular weight S R 47 (P< 0.05) CV 4-4 β1 Acidic shift SS R 50 (P< 0.01) CV 8-1 No change SS R 48 (P <0.02) CV 1-5 α1 Acidic shift SS R 51 (P <0.01) CV 2-8 α1 Basic shift SS R 47 (P <0.02) CV 2-3 No change SS R 50 (P < 0.01) VV 3-2 α1 Acidic shift SS R 48 (P <0.01) - Table 2
Mutations in colcemid- and vinblastine-resistant cell lines
Cell line Wild-type sequence/mutant tubulin sequence Amino acid change Cmd4 CAG-CTG-GAC-CGA-ATC β D45Y CAG-CTG-TAC-CGA-ATCa CV 1-1 TAT-GGG-GAC-CTC-AAC β D224N CV 1-9 TAT-GGG-AAC-CTC-AAC CV 7-1 GAC-ATC-TGC-TTC-CGC β C211F GAC-ATC-TTC-TTC-CGC CV 4-4 AAC-GTC-AAG-ACC-GCC β K350N AAC-GTC-AAT-ACC-GCC CV 8-1 ACC-ATG-AGC-GGG-GTC β S234N ACC-ATG-AAC-GGG-GTC CV 1-2 GCC-TAC-CAT-GAG-CAG α H283Y CV 1-5, CV 1-7 GCC-TAC-TAT-GAG-CAG CV 2-8 TTC-AGT-GAG-ACA-GGC α E55K TTC-AGT-AAG-ACA-GGC CV 2-3 ACC-ACA-GCC-ATC-GCT α A383V ACC-ACA-GTC-ATC-GCT VV 3-2 TGG-GCT-CGC-CTA-GAT α R390C TGG-GCT-TGC-CTA-GAT -
↵a Nucleotides in bold represent the mutations found in each of the mutant cell lines.
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Volume 2, Issue 7
