Elevated levels of microtubule destabilizing factors in a Taxol-resistant/dependent A549 cell line with an alpha-tubulin mutation.
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.
Martello
Laura A
LA
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Verdier-Pinard
Pascal
P
Shen
Heng-Jia
HJ
He
Lifeng
L
Torres
Keila
K
Orr
George A
GA
Horwitz
Susan Band
SB
D000595:Amino Acid Sequence; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D006801:Humans; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis*; D008870:Microtubules / Q000502:physiology; D008969:Molecular Sequence Data; D017239:Paclitaxel / Q000494:pharmacology*; D010750:Phosphoproteins / Q000096:biosynthesis*; D017354:Point Mutation; D011487:Protein Conformation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050777:Stathmin; D014404:Tubulin / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D010750:Phosphoproteins; C493995:STMN1 protein, human; D050777:Stathmin; D014404:Tubulin; D017239:Paclitaxel