title: High-affinity accumulation of a maytansinoid in cells via weak tubulin interaction.
abstract: The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4-6 × 10(7) copies). Efflux of 3[H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of 3 [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.
authors: Department of Cell Biology, ImmunoGen, Inc., Waltham, Massachusetts, United States of America.
authors: Victor S
authors: VS
authors: Goldmacher
authors: Department of Cell Biology, ImmunoGen, Inc., Waltham, Massachusetts, United States of America.
authors: Charlene A
authors: CA
authors: Audette
authors: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America; Renal Division, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors: Yinghua
authors: Y
authors: Guan
authors: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America; Renal Division, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors: Eriene-Heidi
authors: EH
authors: Sidhom
authors: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America; Renal Division, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors: Jagesh V
authors: JV
authors: Shah
authors: Department of Cell Biology, ImmunoGen, Inc., Waltham, Massachusetts, United States of America.
authors: Kathleen R
authors: KR
authors: Whiteman
authors: Department of Cell Biology, ImmunoGen, Inc., Waltham, Massachusetts, United States of America.
authors: Yelena V
authors: YV
authors: Kovtun
mesh_terms: D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008081:Liposomes; D008453:Maytansine / Q000378:metabolism* / Q000494:pharmacology; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D011485:Protein Binding; D014404:Tubulin / Q000378:metabolism*
chemical_list: D008081:Liposomes; D014404:Tubulin; D008453:Maytansine