title: Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of beta-tubulin (Asp26Glu) and less stable microtubules.
abstract: Resistance to paclitaxel-based therapy is frequently encountered in the clinic. The mechanisms of intrinsic or acquired paclitaxel resistance are not well understood. We sought to characterize the resistance mechanisms that develop upon chronic exposure of a cancer cell line to paclitaxel in the presence of the P-glycoprotein reversal agent, CL-347099. The epidermoid tumor line KB-3-1 was exposed to increasing concentrations of paclitaxel and 5 micromol/L CL-347099 for up to 1 year. Cells grown in 15 nmol/L paclitaxel plus CL-347099 (KB-15-PTX/099) developed 18-fold resistance to paclitaxel and were dependent upon paclitaxel for maximal growth. They grew well and retained resistance to paclitaxel when grown in athymic mice. Cross-resistance (3- to 5-fold) was observed in tissue culture to docetaxel, the novel taxane MAC-321, and epothilone B. Collateral sensitivity (approximately 3-fold) was observed to the depolymerizing agents vinblastine, dolastatin-10, and HTI-286. KB-15-PTX/099-resistant cells did not overexpress P-glycoprotein nor did they have an alteration of [14C]paclitaxel accumulation compared with parental cells. However, a novel point mutation (T to A) resulting in Asp26 to glutamate substitution in class I (M40) beta-tubulin was found. Based on an electron crystallography structure of Zn-stabilized tubulin sheets, the phenyl ring of C-3' NHCO-C6H5 of paclitaxel makes contact with Asp26 of beta-tubulin, suggesting a ligand-induced mutation. Optimized model complexes of paclitaxel, docetaxel, and MAC-321 in beta-tubulin show a novel hydrogen bonding pattern for the glutamate mutant and rationalize the observed resistance profiles. However, a mutation in the paclitaxel binding pocket does not explain the phenotype completely. KB-15-PTX/099 cells have impaired microtubule stability as determined by a reduced percentage of tubulin in microtubules and reflected by less acetylated tubulin. These results suggest that a mutation in tubulin might affect microtubule stability as well as drug binding and contribute to the observed resistance profile.
authors.affiliation: Discovery Oncology, Wyeth, 401 North Middletown Road, Room 4709, Building 200, Pearl River, NY 10965, USA.
authors.forename: Malathi
authors.initials: M
authors.lastname: Hari
authors.forename: Frank
authors.initials: F
authors.lastname: Loganzo
authors.forename: Tami
authors.initials: T
authors.lastname: Annable
authors.forename: Xingzhi
authors.initials: X
authors.lastname: Tan
authors.forename: Sylvia
authors.initials: S
authors.lastname: Musto
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Morilla
authors.forename: James H
authors.initials: JH
authors.lastname: Nettles
authors.forename: James P
authors.initials: JP
authors.lastname: Snyder
authors.forename: Lee M
authors.initials: LM
authors.lastname: Greenberger
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000627:therapeutic use*; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000235:genetics; D045744:Cell Line, Tumor; D000077143:Docetaxel; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D034261:Epothilones / Q000737:chemistry / Q000627:therapeutic use; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D008870:Microtubules / Q000235:genetics / Q000378:metabolism; D017239:Paclitaxel / Q000031:analogs & derivatives / Q000737:chemistry / Q000627:therapeutic use*; D017354:Point Mutation; D011487:Protein Conformation; D043823:Taxoids / Q000737:chemistry / Q000627:therapeutic use; D014404:Tubulin / Q000737:chemistry / Q000235:genetics*; D014700:Verapamil / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000972:Antineoplastic Agents, Phytogenic; C510004:CL 347099; D034261:Epothilones; C479501:MAC321; D043823:Taxoids; D014404:Tubulin; D000077143:Docetaxel; D001224:Aspartic Acid; D018698:Glutamic Acid; D014700:Verapamil; D017239:Paclitaxel; C093788:epothilone B