Article Figures & Data
Figures
- Fig. 1.
Chemical structures of Taxol and the Epos.
- Fig. 2.
HeLa.EpoB1.8 cells are dependent on either the Epos or the taxanes for growth. In the presence of low nanomolar concentrations of Taxol, Taxotere, Epo A, or Epo B, there is an approximate 1.5- to 2-fold increase in cell growth compared with cells grown in the absence of drug. Discodermolide does not rescue the growth of HeLa.EpoB1.8 cells. Error bars were omitted for clarity. SD did not exceed 5%.
- Fig. 3.
Epo-resistant cell lines do not express the MDR1 gene. Total RNA was prepared from Epo-resistant cells and examined for the expression of the MDR1 gene by RT-PCR. SKVLB1, a cell line that overexpresses the MDR1 gene, was used as a positive control, and the β2M gene was used as a control for the quality of the samples. Lane 1, A549; Lane 2, A549.EpoB40; Lane 3, HeLa; Lane 4, HeLa.EpoA9; Lane 5, HeLa.EpoB1.8; Lane 6, SKOV3; Lane 7, SKVLB1.
- Fig. 4.
Microtubule protein from A549.EpoB40 cells does not polymerize as well as MTP from A549 cells in the presence of Epo B. Cell lysates prepared from A549 and A549.EpoB40 cells were incubated with the indicated concentrations of Epo B at 37°C for 30 min. Microtubule polymers were centrifuged, and the amount of soluble microtubule protein remaining in the supernatant was determined by Western blot analysis and densitometry as an indication of the level of microtubule assembly. Bars, SD.
- Fig. 5.
Modeling of mutations in β-tubulin. A, B292Gln to Glu, B173Pro to Ala, and B422Tyr to Tyr/Cys mutations are located near the M-loop, the GDP binding site, and the COOH terminus of β-tubulin, respectively. B, B292Gln is close to the M-loop and Thr-274, an amino acid residue that forms part of the Taxol/Epo binding pocket (22, 23). C, B173Pro is on the loop forming part of the nucleotide-binding pocket in β-tubulin. Another amino acid on the loop, B171, is also a Pro. D, the H11 and H12 helixes form the external surface of the microtubule polymer and provide binding sites for MAPs and motor proteins. B422Tyr is located on the H12 helix.
Tables
- Table 1
Drug resistance profile of A549.EpoB40 cells.
IC50s were determined after 72 h of incubation with the indicated drugs. The IC50 of A549.EpoB40 cells was divided by the IC50 of A549 cells to indicate the fold decrease or increase in drug sensitivity.
Compound IC50 (nm) Fold A549 A549.EpoB40 Epo B 0.96 ± 0.11 91.50 ± 3.88 95 Epo A 1.89 ± 0.26 136.91 ± 4.71 72 Taxol 3.12 ± 0.36 67.96 ± 15.40 22 Taxotere 1.34 ± 0.39 16.87 ± 3.91 13 Discoa 7.68 ± 0.64 16.60 ± 2.54 2 VBL 2.31 ± 0.08 1.26 ± 0.47 0.5 CLC 16.86 ± 0.63 10.24 ± 1.03 0.6 -
↵a Disco, discodermolide; VBL, vinblastine; CLC, colchicine.
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- Table 2
Drug resistance profile of HeLa.EpoA9 and HeLa.EpoB1.8 cells.
IC50s were determined after 72 h of incubation with the indicated drugs. The IC50 of HeLa.EpoA9 and HeLa.EpoB1.8 cells were divided by the IC50 of HeLa cells to indicate the fold decrease or increase in drug sensitivity.
Compound HeLa HeLa.EpoA9 HeLa.EpoB1.8 IC50 (nm) IC50 (nm) Fold IC50 (nm) Fold Epo A 4.02 ± 0.01 9.95 ± 0.19 2.5 6.18 ± 0.04 1.5 EpoB 1.96 ± 0.48 3.40 ± 0.08 1.7 4.41 ± 0.16 2.3 Taxol 1.68 ± 0.01 10.70 ± 0.84 6.4 4.75 ± 0.03 2.8 Taxotere 1.42 ± 0.05 7.84 ± 0.15 5.5 4.89 ± 0.06 3.4 Discoa 7.54 ± 0.19 16.01 ± 0.46 2.1 6.70 ± 0.92 0.9 VBL 0.95 ± 0.08 0.81 ± 0.05 0.9 1.55 ± 0.07 1.6 CLC 10.80 ± 0.78 6.24 ± 0.10 0.6 4.52 ± 0.18 0.4 -
↵a Disco, discodermolide; VBL, vinblastine; CLC, colchicine.
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- Table 3
Doubling time for Epo-sensitive and Epo-resistant cell lines.
Cells (40,000 cells/well) were seeded in a six-well plate and allowed to grow to 80% confluency. The total number of cells in each well was counted, and the length of incubation was used to obtain the doubling time.
Cell line Doubling time (h) Increase (%) A549 23.5 A549.EpoB40 31.8 35 HeLa 19.5 HeLa.EpoA9 21.5 10 HeLa.EpoB1.8 43.3 122 - Table 4
Class I β-tubulin mutations in Epo-resistant cell lines.
Total RNA was prepared from Epo-resistant cell lines and the parental drug-sensitive cell lines. RNA was reverse transcribed to cDNA. The class I β-tubulin gene was amplified by PCR and sequenced with four pairs of primers. A mutation is noted after comparison to both the published sequence and the class I β-tubulin in the drug-sensitive parental cell line.
Cell line Amino acid Codon A549.EpoB40 B292Gln → Glu CAG → GAG HeLa.EpoA9 B173Pro → Ala CCC → GCC HeLa.EpoB1.8 B422Tyr → Tyr/Cys TAT → TAT/TGT
Volume 1, Issue 1
