Elevated levels of microtubule destabilizing factors in a taxol-resistant/dependent A549 cell line with an α-tubulin mutation

• Published in: Cancer research (Chicago, Ill.) Vol. 63; no. 6; pp. 1207 - 1213
• Main Authors: MARTELLO, Laura A, VERDIER-PINARD, Pascal, SHEN, Heng-Jia, LIFENG HE, TORRES, Keila, ORR, George A, HORWITZ, Susan Band
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA American Association for Cancer Research 15.03.2003
• Subjects: Amino Acid Sequence; Antineoplastic Agents, Phytogenic - pharmacology; Biological and medical sciences; DNA Mutational Analysis; Drug Resistance, Neoplasm - genetics; General aspects (metabolism, cell proliferation, established cell line...); Humans; Medical sciences; Microtubule Proteins; Microtubule-Associated Proteins - biosynthesis; Microtubules - physiology; Molecular Sequence Data; Paclitaxel - pharmacology; Phosphoproteins - biosynthesis; Point Mutation; Protein Conformation; Reverse Transcriptase Polymerase Chain Reaction; Stathmin; Tubulin - genetics; Tubulin - metabolism; Tumor cell; Tumor Cells, Cultured; Tumors; Antineoplastic agent; Human; Gene expression; Cell line establishment; In vitro; Resistance; Tubulin; Taxane derivatives; Established cell line; Point mutation; Paclitaxel; Instability; Microtubule associated protein; Microtubule; Tumor cell
• ISSN: 0008-5472; 1538-7445

The A549 Taxol-resistant cell lines, A549-T12 and A549-T24, were isolated in our laboratory, and are dependent on Taxol for normal growth. The microtubules in these cells displayed increased dynamicity in the absence of Taxol. In the present study, a heterozygous point mutation in Kalpha1-tubulin was discovered at alpha379 (Ser to Ser/Arg). Although Taxol binds to beta-tubulin in the microtubule, sequencing of beta-tubulin class I did not reveal any mutations. The expression of the alpha-tubulin mutation was demonstrated using high-resolution isoelectric focusing and two-dimensional gel analysis. Both the wild-type and mutant tubulin were expressed in the Taxol-resistant cell lines. The region of alpha-tubulin that encompasses alpha379 is near the COOH terminus that has been proposed as a site of interaction with microtubule-associated protein (MAP) 4 and stathmin, a tubulin-interacting protein. In the Taxol-resistant cells, the active nonphosphorylated form of stathmin was increased approximately 2-fold, whereas the inactive phosphorylated forms were barely detected. The inactive phosphorylated forms of MAP4 were increased in the A549-T12 and A549-T24 cell lines. We hypothesize that these changes in tubulin/MAPs that result in increased microtubule instability may be related to the alpha-tubulin mutation and are compensated for by the stabilizing properties of Taxol.