title: Effect of an experimental proteasome inhibitor on the cytoskeleton, cytosolic protein turnover, and induction in the neuronal cells in vitro.
abstract: GT-1 murine neuronal cells exposed to an experimental proteasome inhibitor (EPI) for 24h showed increased cell death via a non-apoptotic mechanism, as assessed by TUNEL and DNA fragmentation assays. Immunofluorescence staining demonstrated that EPI induced reorganization and relocation of non-ubiquinated actin microfilaments and microtubules to the perinuclear region in EPI treated cells. Immunohistochemistry analysis also demonstrated that other non-cytoskeletal proteins became ubiquitinated and/or upregulated including ubiquitin and other stress proteins. Perinuclear-centrosomal accumulation of gamma-tubulin and vimentin, key components of aggresomes, was observed in the EPI treated cells. Biochemical analysis indicated that EPI-induced accumulation of ubiquitinated protein aggregates in GT-1 cells was detergent - and mechanical - disruption resistant, a feature of aggresomes. Similar results were observed in GT-1 cells treated with lactacystin, a prototypical proteasome inhibitor, which is structurally dissimilar to EPI indicating a pharmacologic effect. In conclusion, EPI causes cytoskeletal reorganization and accumulation of diverse ubiquitinated and non-ubiquitinated proteins in the perinuclear region and potentially overloads the endoplasmic reticulum-dependent quality control mechanism. These processes acting alone, or in combination, are hypothesized to affect axonal transport or other aspects of cellular homeostasis and thus, represent events potentially relevant to the development of peripheral neuropathy associated with administration of proteasome inhibitors in nonclinical studies.
authors.affiliation: Department of Drug Safety Evaluation, Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA. vcsizmad@mpi.com
authors.forename: Vilmos
authors.initials: V
authors.lastname: Csizmadia
authors.forename: Arek
authors.initials: A
authors.lastname: Raczynski
authors.forename: Eva
authors.initials: E
authors.lastname: Csizmadia
authors.forename: Eric R
authors.initials: ER
authors.lastname: Fedyk
authors.forename: James
authors.initials: J
authors.lastname: Rottman
authors.forename: Carl L
authors.initials: CL
authors.lastname: Alden
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000633:toxicity; D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000633:toxicity*; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D006360:Heat-Shock Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D051379:Mice; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D000199:Actins; D015853:Cysteine Proteinase Inhibitors; D006360:Heat-Shock Proteins; D009419:Nerve Tissue Proteins; D061988:Proteasome Inhibitors; D014404:Tubulin; D025801:Ubiquitin; C067713:lactacystin; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine