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Elevated Levels of Microtubule Destabilizing Factors in a Taxol-resistant/dependent A549 Cell Line with an -Tubulin Mutation¹

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461

The A549 Taxol-resistant cell lines, A549-T12 and A549-T24, were isolated in our laboratory, and aredependent on Taxol for normal growth. The microtubules inthese cells displayed increased dynamicity in the absence of Taxol. In the present study, a heterozygous point mutation in K1-tubulin was discovered at 379 (Ser to Ser/Arg). Although Taxol binds to ß-tubulin in the microtubule, sequencing of ß-tubulin class I did not reveal any mutations. The expression of the -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 -tubulin that encompasses 379 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 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 -tubulin mutation and are compensated for by the stabilizing properties of Taxol.

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