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 called ensconsin (E-MAP-115) and degree of Taxol sensitivity in several cultured cell lines prompted us to investigate potential cause-and-effect relationships between ensconsin level and Taxol action. We used human MCF-7 or HeLa cells, which are sensitive to low Taxol concentrations (LD(50) of 30-35 and 3.5 nM, respectively) to prepare stably transfected populations of cells expressing heterogeneous levels of ensconsin chimeras, either green fluorescent protein (GFP) conjugated to full-length ensconsin (GFP-Ensc) or to ensconsin's microtubule-binding domain (GFP-EMTB). Both a subjective microscopic assay, i.e., scoring fluorescence of GFP-ensconsin chimeras following Taxol treatment, and a quantitative immunobiochemical assay, i.e., measuring level of GFP-ensconsin chimera in cells surviving treatment with Taxol, showed that cells expressing higher levels of GFP-ensconsin chimera were killed more readily by Taxol concentrations approaching the LD(50). In contrast, in TC-7 cells, which are relatively insensitive to Taxol (LD(50) > 600 nM), high-level expression of GFP-EMTB conferred no significant susceptibility to killing by Taxol. However, heightening the Taxol sensitivity of GFP-EMTB-TC-7 cells by pre-incubating cells with the p-glycoprotein inhibitor, verapamil, did result in selective killing of cells highly expressing GFP-EMTB. Taken together, results obtained in MCF-7, HeLa, and TC-7 cells suggest that elevated ensconsin level bestowed a selective disadvantage upon Taxol-sensitive cells. To probe potential mechanisms by which ensconsin could alter the Taxol response, we isolated microtubules from HeLa cells that were or were not pretreated with Taxol. In vivo Taxol treatment significantly tightened microtubule-binding of ensconsin, suggesting that Taxol alters ensconsin's microtubule-binding properties and may, in turn, alter the Taxol response of the microtubules. Our data support the hypothesis that Taxol works synergistically or in concert with microtubule-binding proteins in bringing about deleterious effects on the microtubule cytoskeleton.
Gruber
D
D
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Faire
K
K
Bulinski
J C
JC
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D004354:Drug Screening Assays, Antitumor; D004357:Drug Synergism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000032:analysis; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D010080:Oxazoles; D017239:Paclitaxel / Q000494:pharmacology*; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013045:Species Specificity; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology
D000970:Antineoplastic Agents; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D010080:Oxazoles; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C006171:2-(2,6-xylidino)-2-oxazoline; D014747:Vinblastine; D014700:Verapamil; D017239:Paclitaxel