TY - JOUR
T1 - Elevated levels of microtubule destabilizing factors in a Taxol-resistant/dependent A549 cell line with an α-tubulin mutation
AU - Martello, Laura A.
AU - Verdier-Pinard, Pascal
AU - Shen, Heng Jia
AU - He, Lifeng
AU - Torres, Keila
AU - Orr, George A.
AU - Horwitz, Susan Band
PY - 2003/3/15
Y1 - 2003/3/15
N2 - 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 Kα1-tubulin was discovered at α379 (Ser to Ser/Arg). Although Taxol binds to β-tubulin in the microtubule, sequencing of β-tubulin class I did not reveal any mutations. The expression of the α-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 α-tubulin that encompasses α379 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 non-phosphorylated 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.
AB - 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 Kα1-tubulin was discovered at α379 (Ser to Ser/Arg). Although Taxol binds to β-tubulin in the microtubule, sequencing of β-tubulin class I did not reveal any mutations. The expression of the α-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 α-tubulin that encompasses α379 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 non-phosphorylated 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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M3 - Article
C2 - 12649178
AN - SCOPUS:0037444386
SN - 0008-5472
VL - 63
SP - 1207
EP - 1213
JO - Cancer Research
JF - Cancer Research
IS - 6
ER -