Elucidation of apoptosis induced by serum deprivation in cultured conjunctival epithelial cells.
OBJECTIVE
The conjunctival epithelial cell line, CCL20.2 (CCL), requires the presence of 10% fetal calf serum (FCS) in the medium to survive. To elucidate the molecular mechanism underlying such cell death, including the death signal for these cells, the activities of several caspases in the CCL were measured, and the effects of caspase inhibitors and serum components on cell death were examined.


METHODS
CCL was grown in Medium 199 containing 10% FCS, and the medium was changed to Medium 199 with or without 10% FCS, or medium without 10% FCS but containing caspase inhibitors or serum components. After 24 hours' incubation, the enzyme activities of caspases 1, 3, 8, and 9 in the culture supernatants were measured, and the effects of caspase inhibitors and serum components-for example, growth factors, lactoferrin, retinoic acid, were investigated.


RESULTS
DNA fragmentation was induced by serum deprivation, confirming that serum deprivation induces apoptosis in CCL. While the activities of caspases 3 and 8 were found to be increased, those of caspases 1 and 9 were not detected in the apoptotic cells. Z-VAD completely suppressed the caspase 3 activation, and specific inhibitors of caspases 1, 8, and 9 partially suppressed the activation. Serum deprivation induced a decrease in the cellular viability, which, however, partially recovered in the presence of caspase inhibitors, epidermal growth factor and retinoic acid.


CONCLUSIONS
These results suggest that the apoptosis induced by serum deprivation involves caspases 1, 3, 8, and 9, and is suppressed by caspase inhibitors. EGF and retinoic acid have a key role in the maintenance of the ocular surface.
Higuchi
A
A
PhD, 6N9 Research Park, Keio University, School of Medicine, 35 Shinano-Machi, Shinjyuku-Ku, Tokyo, 160-8582 Japan. ahiguchi@sc.itc.keio.ac.jp
Shimmura
S
S
Takeuchi
T
T
Suematsu
M
M
Tsubota
K
K
D017209:Apoptosis / Q000187:drug effects; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism / Q000502:physiology; D002460:Cell Line; D003228:Conjunctiva / Q000201:enzymology / Q000473:pathology*; D016895:Culture Media, Serum-Free; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004847:Epithelial Cells / Q000201:enzymology / Q000473:pathology; D006801:Humans; D013666:Tears / Q000737:chemistry; D014212:Tretinoin / Q000494:pharmacology
D061945:Caspase Inhibitors; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D014212:Tretinoin; D004815:Epidermal Growth Factor; D020169:Caspases