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.2005 Dec;4(12):1847-53.
doi: 10.4161/cc.4.12.2264. Epub 2005 Dec 18.

Resistance to microtubule-stabilizing drugs involves two events: beta-tubulin mutation in one allele followed by loss of the second allele

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Resistance to microtubule-stabilizing drugs involves two events: beta-tubulin mutation in one allele followed by loss of the second allele

Yuefang Wang et al. Cell Cycle.2005 Dec.

Abstract

Resistance to Taxol (paclitaxel) or the epothilones (Epo) occurs via the acquisition of point mutations in beta-tubulin residues important for drug-tubulin binding. We have isolated four drug-resistant clones selected with Taxol or Epo A, which harbor distinct beta-tubulin mutations. During the development of a stable drug-resistant phenotype, early clones expressing both wild-type (wt) and mutant beta-tubulin sequences exhibited a 10-fold drug resistance, while more advanced clones expressing only the mutant beta-tubulin sequence exhibited 30 to 50-fold drug resistance. The drug-sensitive parental 1A9 ovarian carcinoma cell line and the drug resistant clones (1A9-A8, 1A9-PTX10 and 1A9-PTX22) were evaluated for loss of heterozygosity (LOH) for beta-tubulin (6p25) by single nucleotide polymorphism (SNP) and fluorescent in situ hybridization (FISH) analyses. Functional assays such as drug-induced tubulin polymerization, cell cycle analysis by FACS, DNA sequencing for beta-tubulin and mitotic index by immunofluorescence were performed to correlate the beta-tubulin LOH status with drug response in the early- and late-step drug-resistant clones. Late-step drug resistant clones revealed LOH in one allele for wt beta-tubulin in addition to a beta-tubulin mutation in the other allele leading to increased levels of drug resistance, while the early-step clones that contained both a wt and a mutant beta-tubulin allele were considerably less drug resistant. The LOH and functional assays revealed cell response that was proportional to the tubulin gene and heterozygosity status. Acquired tubulin mutations in conjunction with LOH for the wt tubulin resulted in a highly resistant phenotype, revealing a new mechanism for taxane resistance.

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Figures

Figure 1
Figure 1
Sequence analysis of β-tubulin M40 cDNA from 1A9 parental and Epo A resistant cells. A portion of the sequence chromatogram of β-tubulin M40 cDNA exon 4 from 1A9 parental and both EpoR cell lines is shown. The 1A9 parental cell line displays wild type sequence for the M40 β-tubulin amino acid Thr274 (ACC) (top panel), while a homozygous point mutation at this residue β274 (ThrACC to IleATC) is seen in the late-step Epo-resistant clone 1A9-A8 (middle panel). A heterozygous point mutation for the same residue β274 (ThrACC to ThrACC/IleATC) is observed in the early-step Epo-resistant clone 1A9-A8E (lower panel).
Figure 2
Figure 2
1A9-A8 and 1A9-A8E cells exhibit impaired in vivo drug-induced tubulin polymerization compared with their parental 1A9 cells. Drug-sensitive parental 1A9 (A) and the Epo AR clones, 1A9-A8 and 1A9-A8E (B), were treated for 5 h with or without (0) various concentrations of Epo A as indicated. After cell lysis, the polymerized (P) and the soluble (S) protein fractions were separated by centrifugation, resolved by SDS/PAGE, and immunoblotted with an antibody against alpha-tubulin. The percent of polymerized tubulin (%P) was determined by dividing the densitometric value of polymerized tubulin by the total tubulin content (the sum of P plus S). The results shown are from a representative experiment of four independent observations.
Figure 3
Figure 3
Impaired Epothilone-induced G2/M arrest in the 1A9-Epo Cell cycle analysis by flow cytometry was performed in the parental 1A9 and the 1A9-EpoR clones, following overnight treatment with Epo A or Vincristine as indicated. The parental 1A9 cells readily arrested in G2/M after treatment with either microtubule-stabilizing or destabilizing agents. The early step isolate 1A9-A8E was partially arrested in G2/M following Epo A treatment; while the late-step isolate 1A9-A8 failed to arrest in mitosis after treatment with Epo A even at the highest concentration. Both EpoR clones were arrested in G2/M after treatment with 10 nM Vincristine. 10000 events were recorded for each condition, the histogram is representative of three independent experiments.
Figure 4
Figure 4
M40, the major isotype of β-tubulin in human cell, is located on 6p25. (A) PCR amplification from genomic DNA isolated from the BAC clones RP11-527J5 BAC (located at 6p21.3) and RP11-506K6 BAC (located at 6p25), using primers specific for M40 and β9 tubulin isotypes. The primers were designed from intron 3 to the 3’UTR region of each gene, thereby amplifying the entire exon 4. Upon bacterial expansion of the two BACs, five different clones were picked to ensure that the BACs we had received were uniform and homogeneous. As a positive control we used genomic DNA from 1A9 cells. (B) Metaphase spreads from 1A9 cells were hybridized with BAC clones RP11-527J5 (6p21.3) and RP11-506k6 (6p25) shown in orange, and a centromeric probe for chromosome 6 (green), followed by counterstaining with nucleic acid stain Sytox Blue (blue).
Figure 5
Figure 5
SNP marker analysis of 1A9 and 1A9-Resistant Cells. Left Panel: Diagram of chromosome 6p displaying the location of the SNP markers within the 9.5 Mb contig NT_003488. The β-tubulin gene M40 is highlighted within the BAC clone RP11-506k6 located within this contig at 6p25. The location of the four informative SNP markers is displayed. Right Panel: table showing the corresponding SNP nucleotides by DNA sequencing analysis in the parental 1A9 cells, and the four drug-resistant clones, as indicated.
Figure 6
Figure 6
FISH analysis of 1A9, 1A9-A8E and 1A9-A8 cells. Metaphase spreads from all three cell lines were hybridized with BAC clone RP11-506k6 (orange) containing the β-tubulin gene M40, and a centromeric probe for chromosome 6 (green), followed by counterstaining with nucleic acid stain Sytox Blue (blue). The parental 1A9 cells display two copies of chromosome 6 (white arrows) as evidenced by the chromosome 6 centromeric probe staining. Both 6 chromosomes displayed staining for the BAC clone indicating two copies of the β-tubulin gene M40. The early-step clone 1A9-A8E presented a similar karyotype as the parental cells, with two copies of chromosome 6 each containing the β-tubulin BAC clone. In the late-step 1A9-A8 cells however, only one chromosome 6 stained for the BAC clone, although both copies of the chromosome 6 were present (left panel). The white arrows indicate chromosomes 6, as evidenced by the green centromeric staining, and the BAC hybridization at the tips of the chromosome. In 1A9-A8 the yellow dashed arrow indicates the chromosome 6 that has lost the chromosomal region of 6p25. Insets display either a magnification of chromosome 6 in metaphase, or interphase. DNA in interphase cells and individual chromosomes in the metaphase plate are stained in blue. Scale bar indicates 5 μm.
Figure 7
Figure 7
Temporal model for the development of high level of drug resistance to Epothilone A. Upon selection with Epothilone A a single point mutation occurs in one β-tubulin allele producing a low resistance profile in the cell. During continued selection with the drug, loss of heterozygosity of the remaining wild type allele of β-tubulin occurs along with duplication of the mutant allele generating a high resistance clone.
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