TY - JOUR
T1 - Paclitaxel-resistant human ovarian cancer cells have mutant β-tubulins that exhibit impaired paclitaxel-driven polymerization
AU - Giannakakou, Paraskevi
AU - Sackett, Dan L.
AU - Kang, Yoon Koo
AU - Zhan, Zhirong
AU - Buters, Jeroen T.M.
AU - Fojo, Tito
AU - Poruchynsky, Marianne S.
PY - 1997/7/4
Y1 - 1997/7/4
N2 - Acquired resistance to paclitaxel can be mediated by P-glycoprotein or by alterations involving tubulin. We report two paclitaxel-resistant sublines derived from 1A9 human ovarian carcinoma cells. Single-step paclitaxel selection with verapamil yielded two clones that are resistant to paclitaxel and collaterally sensitive to vinblastine. The resistant sublines are not paclitaxel-dependent, and resistance remained stable after 3 years of drug- free culture. All cell lines accumulate [3H]paclitaxel equally, and no MDR- 1 mRNA was detected by polymerase chain reaction following reverse transcription. Total tubulin content is similar, but the polymerized fraction increased in parental but not in resistant cells following the paclitaxel addition. Purified tubulin from parental cells demonstrated paclitaxel- driven increased polymerization, in contrast to resistant cell tubulin, which did not polymerize under identical conditions. In contrast, epothilone B, an agent to which the resistant cells retained sensitivity, increased assembly. Comparable expression of β-tubulin isotypes was found in parental and resistant cells, with predominant expression of the M40 and β2 isotypes. Sequence analysis demonstrated acquired mutations in the M40 isotype at nucleotide 810 (T → G; Phe270 → Val) in 1A9PTX10 cells and nucleotide 1092 (G → A; Ala364 → Thr) in 1A9PTX22 cells. These results identify residues β270 and β364 as important modulators of paclitaxel's interaction with tubulin.
AB - Acquired resistance to paclitaxel can be mediated by P-glycoprotein or by alterations involving tubulin. We report two paclitaxel-resistant sublines derived from 1A9 human ovarian carcinoma cells. Single-step paclitaxel selection with verapamil yielded two clones that are resistant to paclitaxel and collaterally sensitive to vinblastine. The resistant sublines are not paclitaxel-dependent, and resistance remained stable after 3 years of drug- free culture. All cell lines accumulate [3H]paclitaxel equally, and no MDR- 1 mRNA was detected by polymerase chain reaction following reverse transcription. Total tubulin content is similar, but the polymerized fraction increased in parental but not in resistant cells following the paclitaxel addition. Purified tubulin from parental cells demonstrated paclitaxel- driven increased polymerization, in contrast to resistant cell tubulin, which did not polymerize under identical conditions. In contrast, epothilone B, an agent to which the resistant cells retained sensitivity, increased assembly. Comparable expression of β-tubulin isotypes was found in parental and resistant cells, with predominant expression of the M40 and β2 isotypes. Sequence analysis demonstrated acquired mutations in the M40 isotype at nucleotide 810 (T → G; Phe270 → Val) in 1A9PTX10 cells and nucleotide 1092 (G → A; Ala364 → Thr) in 1A9PTX22 cells. These results identify residues β270 and β364 as important modulators of paclitaxel's interaction with tubulin.
UR - http://www.scopus.com/inward/record.url?scp=0030758777&partnerID=8YFLogxK
U2 - 10.1074/jbc.272.27.17118
DO - 10.1074/jbc.272.27.17118
M3 - Article
C2 - 9202030
AN - SCOPUS:0030758777
SN - 0021-9258
VL - 272
SP - 17118
EP - 17125
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 27
ER -