title: Nocodazole Induced Suicidal Death of Human Erythrocytes.
abstract: BACKGROUND
  The microtubule assembly inhibitor nocodazole has been shown to trigger caspase-independent mitotic death and caspase dependent apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether and how nocodazole induces eryptosis.
  
  
  METHODS
  Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, the abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence as well as ceramide surface abundance utilizing specific antibodies. Tubulin abundance was quantified by TubulinTracker™ Green reagent and visualized by confocal microscopy.
  
  
  RESULTS
  A 48 hours exposure of human erythrocytes to nocodazole (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying average forward scatter. Nocodazole significantly increased Fluo3-fluorescence, significantly increased DCF fluorescence and significantly increased ceramide surface abundance. The effect of nocodazole on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+ and was not modified in the presence of Caspase 3 inhibitor zVAD (1 µM). Nocodazole treatment reduced the content of total tubulin.
  
  
  CONCLUSIONS
  Nocodazole triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry, oxidative stress and ceramide.
authors.affiliation: Department of Physiology, University of Tx00FC;bingen, Tuebingen, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Signoretto
authors.forename: Sabina
authors.initials: S
authors.lastname: Honisch
authors.forename: Marilena
authors.initials: M
authors.lastname: Briglia
authors.forename: Caterina
authors.initials: C
authors.lastname: Faggio
authors.forename: Michela
authors.initials: M
authors.lastname: Castagna
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000187:drug effects; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D018613:Microscopy, Confocal; D015739:Nocodazole / Q000633:toxicity*; D010718:Phosphatidylserines / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D050257:Tubulin Modulators / Q000633:toxicity*; D014966:Xanthenes / Q000737:chemistry
chemical_list: D000814:Aniline Compounds; D002518:Ceramides; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D014404:Tubulin; D050257:Tubulin Modulators; D014966:Xanthenes; C059715:Fluo-3; D015739:Nocodazole; D002118:Calcium