Estramustine binds a MAP-1-like protein to inhibit microtubule assembly in vitro and disrupt microtubule organization in DU 145 cells.
The twofold purpose of the study was (a) to determine if a MAP-1-like protein was expressed in human prostatic DU 145 cells and (b) to demonstrate whether a novel antimicrotubule drug, estramustine, binds the MAP-1-like protein to disrupt microtubules. SDS-PAGE and Western blots showed that a 330-kD protein was associated with microtubules isolated in an assembly buffer containing 10 microM taxol and 10 mM adenylylimidodiphosphate. After purification to homogeneity on an A5m agarose column, the 330-kD protein was found to promote 6 S tubulin assembly. Turbidimetric (A350), SDS-PAGE, and electron microscopic studies revealed that micromolar estramustine inhibited assembly promoted by the 330-kD protein. Similarly, estramustine inhibited binding of the 330-kD protein to 6-S microtubules independently stimulated to assemble with taxol. Immunofluorescent studies with beta-tubulin antibody (27B) and MAP-1 antibody (MI-AI) revealed that 60 microM estramustine (a) caused disassembly of MAP-1 microtubules in DU 145 cells and (b) removed MAP-1 from the surfaces of microtubules stabilized with 0.1 microM taxol. Taken together the data suggested that estramustine binds to a 330-kD MAP-1-like protein to disrupt microtubules in tumor cells.
Stearns
M E
ME
Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Wang
M
M
Tew
K D
KD
Binder
L I
LI
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004961:Estramustine / Q000378:metabolism* / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000378:metabolism*; D008870:Microtubules / Q000032:analysis / Q000187:drug effects / Q000648:ultrastructure*; D009588:Nitrogen Mustard Compounds / Q000378:metabolism*; D011471:Prostatic Neoplasms; D014407:Tumor Cells, Cultured
D008869:Microtubule-Associated Proteins; D009588:Nitrogen Mustard Compounds; D004961:Estramustine