This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Yang, C.-P. H. Articles by Horwitz, S. B. Search for Related Content PubMed PubMed Citation Articles by Yang, C.-P. H. Articles by Horwitz, S. B.

¹ Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York; ² Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York; and ³ Department of Natural Product Chemistry, Geselleschaft fur Biotechnologische Forschung, Braunschweig, Germany

Request for reprints: Susan B. Horwitz, Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718-430-2163; Fax: 718-430-8959. E-mail: shorwitz{at}aecom.yu.edu

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 ßI-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 ß-tubulin and K1-tubulin genes in EpoB480 showed that, in addition to the ß292 mutation, ß60 was mutated from Val to Phe and 195 was mutated from Leu to Met. Mass spectrometry indicated that both the Val⁶⁰Phe and Leu¹⁹⁵Met mutations in ßI- and K1-tubulin, respectively, were expressed at the protein level. Molecular modeling indicated that ß60 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 ß60 could inhibit the lateral contacts between protofilaments, thereby destabilizing microtubules. 195 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 195 could modulate the interactions between tubulin and regulatory proteins. We propose that the ßVal⁶⁰Phe mutation plays a critical role in the drug-dependent phenotype of EpoB480 cells.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1/19/05; revised 3/ 8/05; accepted 4/12/05.