A highly epothilone B-resistant A549 cell line with mutations in tubulin that confer drug dependence

Chia-Ping H. Yang, Pascal Verdier-Pinard, Fang Wang, Eva Lippaine-Horvath, Lifeng He, Dansu Li, Gerhard Höfle, Iwao Ojima, George A. Orr, Susan Band Horwitz

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

A 95-fold epothilone B (EpoB)-resistant, but not dependent, A549 human lung carcinoma cell line, A549.EpoB40 (EpoB40), has a Gln to Glu mutation at residue 292 that is situated near the M-loop of βI-tubulin. Further selection of this cell line with higher concentrations of EpoB produced A549.EpoB480 (EpoB480), which is ∼900-fold resistant to EpoB. This cell line, like EpoB40, exhibits cross-resistance to Taxol and extreme sensitivity to vinblastine, but in contrast to EpoB40 it is unusually dependent on EpoB, requiring a minimum of 125 nmol/L EpoB to maintain normal growth. Sequence analysis of the β-tubulin and Kα1-tubulin genes in EpoB480 showed that, in addition to the β292 mutation, β60 was mutated from Val to Phe and α195 was mutated from Leu to Met. Mass spectrometry indicated that both the Val60Phe and Leu195Met mutations in βI- and Kα1-tubulin, respectively, were expressed at the protein level. Molecular modeling indicated that β60 is located at the end of the H1-S2 loop that has been implicated as a principal partner of the M-loop for contacts between protofilaments. A mutation at β60 could inhibit the lateral contacts between proto-filaments, thereby destabilizing microtubules. α195 is located at the external surface of the microtubule that has been proposed as the domain that interacts with a variety of endogenous proteins, such as stathmin and microtubule-associated protein 4. A mutation at α195 could modulate the interactions between tubulin and regulatory proteins. We propose that the βVal60Phe mutation plays a critical role in the drug-dependent phenotype of EpoB480 cells.

Original languageEnglish (US)
Pages (from-to)987-995
Number of pages9
JournalMolecular Cancer Therapeutics
Volume4
Issue number6
DOIs
StatePublished - Jun 2005

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Tubulin
Substance-Related Disorders
Mutation
Microtubules
Stathmin
Cell Line
Proteins
Vinblastine
Paclitaxel
Sequence Analysis
A549 Cells
epothilone B
Mass Spectrometry
Carcinoma
Phenotype
Lung
Growth
Pharmaceutical Preparations
Genes

ASJC Scopus subject areas

  • Oncology
  • Drug Discovery
  • Pharmacology

Cite this

A highly epothilone B-resistant A549 cell line with mutations in tubulin that confer drug dependence. / Yang, Chia-Ping H.; Verdier-Pinard, Pascal; Wang, Fang; Lippaine-Horvath, Eva; He, Lifeng; Li, Dansu; Höfle, Gerhard; Ojima, Iwao; Orr, George A.; Band Horwitz, Susan.

In: Molecular Cancer Therapeutics, Vol. 4, No. 6, 06.2005, p. 987-995.

Research output: Contribution to journalArticle

Yang, Chia-Ping H. ; Verdier-Pinard, Pascal ; Wang, Fang ; Lippaine-Horvath, Eva ; He, Lifeng ; Li, Dansu ; Höfle, Gerhard ; Ojima, Iwao ; Orr, George A. ; Band Horwitz, Susan. / A highly epothilone B-resistant A549 cell line with mutations in tubulin that confer drug dependence. In: Molecular Cancer Therapeutics. 2005 ; Vol. 4, No. 6. pp. 987-995.
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abstract = "A 95-fold epothilone B (EpoB)-resistant, but not dependent, A549 human lung carcinoma cell line, A549.EpoB40 (EpoB40), has a Gln to Glu mutation at residue 292 that is situated near the M-loop of βI-tubulin. Further selection of this cell line with higher concentrations of EpoB produced A549.EpoB480 (EpoB480), which is ∼900-fold resistant to EpoB. This cell line, like EpoB40, exhibits cross-resistance to Taxol and extreme sensitivity to vinblastine, but in contrast to EpoB40 it is unusually dependent on EpoB, requiring a minimum of 125 nmol/L EpoB to maintain normal growth. Sequence analysis of the β-tubulin and Kα1-tubulin genes in EpoB480 showed that, in addition to the β292 mutation, β60 was mutated from Val to Phe and α195 was mutated from Leu to Met. Mass spectrometry indicated that both the Val60Phe and Leu195Met mutations in βI- and Kα1-tubulin, respectively, were expressed at the protein level. Molecular modeling indicated that β60 is located at the end of the H1-S2 loop that has been implicated as a principal partner of the M-loop for contacts between protofilaments. A mutation at β60 could inhibit the lateral contacts between proto-filaments, thereby destabilizing microtubules. α195 is located at the external surface of the microtubule that has been proposed as the domain that interacts with a variety of endogenous proteins, such as stathmin and microtubule-associated protein 4. A mutation at α195 could modulate the interactions between tubulin and regulatory proteins. We propose that the βVal60Phe mutation plays a critical role in the drug-dependent phenotype of EpoB480 cells.",
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T1 - A highly epothilone B-resistant A549 cell line with mutations in tubulin that confer drug dependence

AU - Yang, Chia-Ping H.

AU - Verdier-Pinard, Pascal

AU - Wang, Fang

AU - Lippaine-Horvath, Eva

AU - He, Lifeng

AU - Li, Dansu

AU - Höfle, Gerhard

AU - Ojima, Iwao

AU - Orr, George A.

AU - Band Horwitz, Susan

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