Elevated levels of microtubule destabilizing factors in a Taxol-resistant/dependent A549 cell line with an alpha-tubulin mutation.

Abstract

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.