Abundant expression of the microtubule-associated protein, ensconsin (E-MAP-115), alters the cellular response to Taxol

Correlation between expression level of a microtubule-associated protein ca lled ensconsin (E-MAP-115) and degree of Taxol sensitivity in several cultu red cell lines prompted us to investigate potential cause-and-effect relati onships between ensconsin level and Taxol action. We used human MCF-7 or He La cells, which are sensitive to low Taxol concentrations (LD50 of 30-35 an d 3.5 nM, respectively) to prepare stably transfected populations of cells expressing heterogeneous levels of ensconsin chimeras, either green fluores cent protein (GFP) conjugated to full-length ensconsin (GFP-Ensc) or to ens consin's microtubule-binding domain (GFP-EMTB). Both a subjective microscop ic assay, i.e., scoring fluorescence of GFP-ensconsin chimeras following Ta xol treatment, and a quantitative immunobiochemical assay, i.e., measuring level of GFP-ensconsin chimera in cells surviving treatment with Taxol, sho wed that cells expressing higher levels of GFP-ensconsin chimera were kille d more readily by Taxol concentrations approaching the LD50. In contrast, i n TC-7 cells, which are relatively insensitive to Taxol (LD50 > 600 nM), hi gh-level expression of GFP-EMTB conferred no significant susceptibility to killing by Taxol. However, heightening the Taxol sensitivity of GFP-ENTTB-T C-7 cells by pre-incubating cells with the p-glycoprotein inhibitor, verapa mil, did result in selective killing of cells highly expressing GFP-EMTB. T aken together, results obtained in MCF-7, HeLa, and TC-7 cells suggest that elevated ensconsin level bestowed a selective disadvantage upon Taxol-sens itive cells. To probe potential mechanisms by which ensconsin could alter t he Taxol response, we isolated microtubules from HeLa cells that were or we re not pretreated with Taxol. In vivo Taxol treatment significantly tighten ed microtubule-binding of ensconsin, suggesting that Taxol alters ensconsin 's microtubule-binding properties and may, in tam, alter the Taxol response of the microtubules. Our data support the hypothesis that Taxol works syne rgistically or in concert with microtubule-binding proteins in bringing abo ut deleterious effects on the microtubule cytoskeleton. (C) 2001 Wiley-Liss , Inc.