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Elucidation of apoptosis induced by serum deprivation in cultured conjunctival epithelial cells
  1. A Higuchi1,
  2. S Shimmura2,
  3. T Takeuchi3,
  4. M Suematsu4,
  5. K Tsubota2
  1. 16N9 Research Park, Keio University, School of Medicine, Tokyo, Japan
  2. 2Department of Ophthalmology, Keio University, School of Medicine, Tokyo, Japan
  3. 3Second Department of Internal Medicine, Saitama Medical School, Saitama, Japan
  4. 4Department of Biochemistry and Integrative Medical Biology, Keio University, School of Medicine, Tokyo, Japan
  1. Correspondence to: Akihiro Higuchi PhD, 6N9 Research Park, Keio University, School of Medicine, 35 Shinano-Machi, Shinjyuku-Ku, Tokyo, 160-8582 Japan; ahiguchi{at}sc.itc.keio.ac.jp

Abstract

Background/aims: 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.

Conclusion: 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.

  • ABTS, 2,2′-azinobis(3-ethylbenzothiazolin-6-sulfonic acid
  • AFC, 7-amino-4-trifluoromethyl coumarin
  • CCL, conjunctival epithelial cell line
  • CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid
  • EGF, epidermal growth factor
  • FCS, fetal calf serum
  • FGF, fibroblast growth factor
  • FMK, fluoromethyl ketone
  • HGF, hepatocyte growth factor
  • IGF, insulin-like growth factor
  • NGF, nerve growth factor
  • PDGF, platelet derived growth factor
  • SCF, stem cell factor
  • TGF-β, transforming growth factor-β
  • Z-LEHD, Z-Leu-Glu-His-Asp-FMK
  • Z-LETD, Z-Leu-Glu-Thr-Asp-FMK
  • Z-WEHD, N-benzyloxycarbonyl-Trp-Glu-His-Asp-FMK
  • Z-VAD, Z-Val-Ala-Asp (O-methyl)-FMK
  • apoptosis
  • caspase
  • cell culture
  • conjunctiva
  • dry eye
  • ABTS, 2,2′-azinobis(3-ethylbenzothiazolin-6-sulfonic acid
  • AFC, 7-amino-4-trifluoromethyl coumarin
  • CCL, conjunctival epithelial cell line
  • CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid
  • EGF, epidermal growth factor
  • FCS, fetal calf serum
  • FGF, fibroblast growth factor
  • FMK, fluoromethyl ketone
  • HGF, hepatocyte growth factor
  • IGF, insulin-like growth factor
  • NGF, nerve growth factor
  • PDGF, platelet derived growth factor
  • SCF, stem cell factor
  • TGF-β, transforming growth factor-β
  • Z-LEHD, Z-Leu-Glu-His-Asp-FMK
  • Z-LETD, Z-Leu-Glu-Thr-Asp-FMK
  • Z-WEHD, N-benzyloxycarbonyl-Trp-Glu-His-Asp-FMK
  • Z-VAD, Z-Val-Ala-Asp (O-methyl)-FMK
  • apoptosis
  • caspase
  • cell culture
  • conjunctiva
  • dry eye

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Footnotes

  • Grant support: Research on Eye and Ear Sciences, Immunology, Allergy and Organ Transplantation.

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