Caspase and proteasome activity during staurosporin-induced apoptosis in lens epithelial cells.
OBJECTIVE
To determine what caspases are activated during staurosporin-induced apoptosis in cultured bovine lens epithelial cells (BLECs), to study the time course of caspase activation in relation to morphologic changes, and to investigate the effect of caspase and/or proteasome inhibition on apoptosis.


METHODS
BLECs were incubated with staurosporin at different concentrations or for different times. Phosphatidylserine (PS) externalization was detected by annexin-V labeling, nuclear morphology was studied by staining with Hoechst 33342 stain (Hoechst, Frankfurt, Germany), and the percentage of apoptotic cells was determined by the TdT-dUTP terminal nick-end labeling (TUNEL) assay. The activity of caspase-1, -2, -3, -4, -8, and -9 as well as the chymotrypsin-like activity of the proteasome was measured by the use of fluorogenic peptide substrates. Inhibition of the proteasome was performed by incubation with 10 microM lactacystin, and caspases were inhibited by 1 microM Z-DEVD-FMK or 20 microM Z-VAD-FMK.


RESULTS
Staurosporin treatment caused a dose- and time-dependent increase in the number of apoptotic cells and in caspase-3 activity. Activation of caspase-2, -4, -8, and -9 was also seen. Caspase activity was increased after 3 hours' incubation with 1 microM staurosporin, which is also the time when most cells became annexin-V-positive. Nuclear changes indicative of apoptosis, viewed with both Hoechst and TUNEL staining, appeared after 4 to 6 hours of staurosporin incubation. Incubation of BLECs with lactacystin caused reduction of proteasome activity and increased apoptosis, evidenced in both the TUNEL assay and caspase-3 activation. Preincubation of lens epithelial cells with caspase inhibitors caused complete inhibition of lactacystin- or staurosporin-induced caspase-3 activation (Z-DEVD-FMK/Z-VAD-FMK) and also of caspase-2, -4, -8, and -9 (Z-VAD-FMK), but the reduction in TUNEL-positive cells was only partial. PS translocation and DNA fragmentation after staurosporin treatment occurred despite complete caspase blockade.


CONCLUSIONS
Staurosporin-induced apoptosis in BLECs involves activation of several caspases. Inhibition of the proteasome causes caspase-3 activation and apoptosis. Both staurosporin- and lactacystin-induced apoptosis can be executed in a caspase-independent manner. The present data are useful for understanding of proteolytic mechanisms during apoptosis in lens epithelial cells, which may be an important event in normal lens development as well as in some types of cataract.
Andersson
M
M
Section of Ophthalmology, Institute of Clinical Neuroscience, Medical Faculty, Göteborg University, Sweden. madeleine.andersson@anatcell.gu.se
Sjöstrand
J
J
Petersen
A
A
Honarvar
A K
AK
Karlsson
J O
JO
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001562:Benzimidazoles; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D005456:Fluorescent Dyes; D020287:In Situ Nick-End Labeling; D007908:Lens, Crystalline / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D019311:Staurosporine / Q000494:pharmacology*; D013997:Time Factors
D000590:Amino Acid Chloromethyl Ketones; D001562:Benzimidazoles; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009097:Multienzyme Complexes; D009842:Oligopeptides; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C067713:lactacystin; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D019311:Staurosporine; C017807:bisbenzimide ethoxide trihydrochloride; D000111:Acetylcysteine