Colchicine-induced cytoskeletal collapse and apoptosis in N-18 neuroblastoma cultures is rapidly reversed by applied S-100beta.
Brain connections depend on a stable association between dendrites and axons whose cytoskeleton is stabilized by the proteins MAP-2 and tau, respectively. The glial protein S-100beta inhibits the phosphorylation by PKC of these two microtubule-associated proteins. In order to determine if exogenous S-100beta can directly influence the cytoskeleton of living cells, cultures of N-18 cells (neuroblastoma clonal cell line) are treated for 30 min in serum-free medium with 10(-6) M colchicine. In normal media, colchicine induces a rapid retraction of processes, membrane blebbing, nuclear collapse, and cell death. The observed cellular changes, due to cytoskeletal collapse after exposure to colchicine, are similar and consistent with the loss of processes and cytoplasmic blebbing seen in cells undergoing apoptosis. The addition of 20 ng/ml of S-100beta after the initial 30-min exposure to colchicine prevents apoptosis, nuclear collapse and induces the regrowth of retracted processes. Cells were treated with the Hoechst Stain, a fluorescent marker that binds to nuclear material, to determine the occurrence of apoptosis in our cultures. In our control cultures, receiving no drugs, we found that 15.1% of the cells were apoptotic. When colchicine was added to the culture medium we found that 31.6% of the cells became apoptotic. However, when colchicine was followed by exposure to S-100beta we found that only 5.4% of the cells were apoptotic. Our results suggest that extracellular application of the glial protein S-100beta is sufficient to reverse colchicine-induced cytoskeletal collapse and prevent the resultant apoptosis of the cells. The increased levels of S-100beta seen after brain injury and in certain neurological and psychiatric disorders may be considered as beneficial for brain recovery.
Brewton
L S
LS
Department of Biology, 10-09 Main Building, New York University, 100 Washington Square East, New York, NY 10003, USA.
Haddad
L
L
Azmitia
E C
EC
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001562:Benzimidazoles / Q000493:pharmacokinetics; D002455:Cell Division / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D003078:Colchicine / Q000037:antagonists & inhibitors*; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004347:Drug Interactions / Q000502:physiology; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006801:Humans; D018715:Microscopy, Video; D009410:Nerve Degeneration / Q000188:drug therapy / Q000503:physiopathology / Q000517:prevention & control*; D009414:Nerve Growth Factors; D009416:Nerve Regeneration / Q000187:drug effects* / Q000502:physiology; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology
D001562:Benzimidazoles; D016895:Culture Media, Serum-Free; D005456:Fluorescent Dyes; D009414:Nerve Growth Factors; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C017807:bisbenzimide ethoxide trihydrochloride; D003078:Colchicine