title: Staurosporine-induced apoptosis in human cornea epithelial cells in vitro.
abstract: BACKGROUND
  Apoptosis is a an important process in corneal development, homeostasis, and disease. This study was performed to determine for the first time basic temporal apoptotic features of SV-40 immortalized human corneal epithelial (HCE) cells. Additionally, we introduce a sensitive analysis of confocal microscopic images to measure the kinetics of staurosporine (STS) induced phosphatidylserine (PS) membrane translocation and early nuclear morphological changes.
  
  
  METHODS
  HCE cells were cultured in the presence of STS to induce apoptosis. Caspase-3 activity was measured with the fluorogenic substrate z-DEVD-rhodamine 110. We determined mitochondrial viability with a 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate reduction assay, and chromatin degradation with a fluorometric method using 4,6-diamidino-2-phenylindole (DAPI). Membrane translocation of PS and nuclear alterations were assessed by quantitative fluorescence microscopy. Image processing routines were written in interactive data language (IDL).
  
  
  RESULTS
  Nuclear alterations like hyperchromicity, pyknosis, and active chromatin reorganization evolved instantly after STS induction. They were followed by PS translocation, DNA fragmentation, mitochondrial breakdown, and caspase-3 activation, which were detected between approximately 90 min and 4 h.
  
  
  CONCLUSIONS
  Morphological and texture sensitive descriptors proved to be highly susceptible for the quantification of early apoptotic nuclear characteristics in HCE cells. We propose this method to be considered for the detection of subtle nuclear reorganization in cellular studies.
authors.affiliation: University of Bremen, Institute of Biophysics, Bremen, Germany. shaertel@physik.uni-bremen.de
authors.forename: Steffen
authors.initials: S
authors.lastname: Härtel
authors.forename: Michaela
authors.initials: M
authors.lastname: Zorn-Kruppa
authors.forename: Svitlana
authors.initials: S
authors.lastname: Tykhonova
authors.forename: Päivi
authors.initials: P
authors.lastname: Alajuuma
authors.forename: Maria
authors.initials: M
authors.lastname: Engelke
authors.forename: Horst A
authors.initials: HA
authors.lastname: Diehl
mesh_terms: D017209:Apoptosis; D001692:Biological Transport; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D003315:Cornea / Q000166:cytology*; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D002843:Chromatin; D004791:Enzyme Inhibitors; D010718:Phosphatidylserines; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine