Class I beta-tubulin mutations in 2-methoxyestradiol-resistant acute lymphoblastic leukemia cells: implications for drug-target interactions.
2-Methoxyestradiol (2ME2) is a naturally occurring derivative of estradiol that has been shown to be an active small molecule that has antitumor and antiangiogenic properties. 2ME2 binds to beta-tubulin near the colchicine-binding site, inhibits microtubule polymerization, and induces mitotic arrest. To improve understanding of the mechanisms of action and resistance to 2ME2, we selected leukemia cells, CCRF-CEM, that display increasing resistance to 2ME2, and three of the highly resistant sublines were chosen for detailed analysis. The 2ME2 cells selected in 7.2 to 28.8 micromol/L were found to be 47- to 107-fold resistant to 2ME2 and exhibited low levels of cross-resistance to vinblastine. Two of the lowest 2ME2-resistant sublines were significantly hypersensitive to colchicine and epothilone B, but the hypersensitive effects were lost in the highest 2ME2-resistant subline. Moreover, 2ME2-resistant cells require 10-fold higher concentrations of 2ME2 to induce G(2)-M cell cycle arrest and have higher amounts of tubulin polymer compared with parental cells. Gene and protein sequencing revealed four class I beta-tubulin mutations, S25N, D197N, A248T, and K350N, in the 2ME2-resistant cells. The S25N mutation is within the paclitaxel-binding site, whereas A248T and K350N are within the colchicine-binding site on beta-tubulin, yet the resistant cells were not cross-resistant to paclitaxel or colchicine. This strongly suggests that the mutations have induced conformational changes to the binding site that resulted in 2ME2 resistance. The 2ME2-resistant leukemia cells provide novel insights into microtubule stability and drug-target interactions.
Children's Cancer Institute Australia for Medical Research, P.O. Box 81, Randwick, New South Wales, Australia 2031.
Tracy Y E
TY
Liaw
Noeris K
NK
Salam
Matthew J
MJ
McKay
Anne M
AM
Cunningham
David E
DE
Hibbs
Maria
M
Kavallaris
D000077584:2-Methoxyestradiol; D000107:Acetylation / Q000187:drug effects; D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008938:Mitosis / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics* / Q000473:pathology*; D040681:Structural Homology, Protein; D014404:Tubulin / Q000737:chemistry / Q000235:genetics*
D014404:Tubulin; D004958:Estradiol; D000077584:2-Methoxyestradiol