Staurosporine-induced apoptosis in human cornea epithelial cells in vitro.
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.
Härtel
Steffen
S
University of Bremen, Institute of Biophysics, Bremen, Germany. shaertel@physik.uni-bremen.de
Zorn-Kruppa
Michaela
M
Tykhonova
Svitlana
S
Alajuuma
Päivi
P
Engelke
Maria
M
Diehl
Horst A
HA
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
D002843:Chromatin; D004791:Enzyme Inhibitors; D010718:Phosphatidylserines; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine