Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells

Jiyang Cai, Mei Wu, Kasey C. Nelson, Paul Sternberg, Dean P. Jones

Research output: Contribution to journalArticle

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Abstract

PURPOSE. To determine the mechanism of oxidant-induced cell death in cultured human retinal pigment epithelium (hRPE). METHODS. Cultured hRPE cells were treated with different concentrations of a chemical oxidant, t- butylhydroperoxide (tBH), for different periods of time. Apoptosis was determined with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and flow cytometry. Mitochondrial membrane potential (mtΔΨ) was measured by rhodamine 123 staining and subsequent flow cytometry. Release of mitochondrial cytochrome c (cyt c) and cleavage of procaspase 3 and caspase substrates were determined by western blot analysis. RESULTS. t-Butylhydroperoxide caused time- and dose-dependent activation of apoptosis in hRPE, indicated by characteristic morphologic changes; TUNEL- positive labeling; phosphatidylserine (PS) exposure; and procaspase 3, poly(ADP-ribose)polymerase, lamin, and tubulin cleavage. An early decrease of mtΔΨ was observed before caspase activation, together with the release of mitochondrial cyt c. CONCLUSIONS. Results indicate that tBH can induce apoptosis in hRPE, probably by triggering the mitochondrial permeability transition, which results in swelling and release of mitochondrial intermembrane proteins.

Original languageEnglish (US)
Pages (from-to)959-966
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume40
Issue number5
StatePublished - 1999
Externally publishedYes

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Retinal Pigments
Retinal Pigment Epithelium
tert-Butylhydroperoxide
Oxidants
Epithelial Cells
Apoptosis
Caspases
Cytochromes c
Caspase 3
Flow Cytometry
Lamins
Rhodamine 123
DNA Nucleotidylexotransferase
Poly(ADP-ribose) Polymerases
Mitochondrial Membrane Potential
Mitochondrial Proteins
Phosphatidylserines
Tubulin
Permeability
Cell Death

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Cai, J., Wu, M., Nelson, K. C., Sternberg, P., & Jones, D. P. (1999). Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells. Investigative Ophthalmology and Visual Science, 40(5), 959-966.

Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells. / Cai, Jiyang; Wu, Mei; Nelson, Kasey C.; Sternberg, Paul; Jones, Dean P.

In: Investigative Ophthalmology and Visual Science, Vol. 40, No. 5, 1999, p. 959-966.

Research output: Contribution to journalArticle

Cai, J, Wu, M, Nelson, KC, Sternberg, P & Jones, DP 1999, 'Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells', Investigative Ophthalmology and Visual Science, vol. 40, no. 5, pp. 959-966.
Cai, Jiyang ; Wu, Mei ; Nelson, Kasey C. ; Sternberg, Paul ; Jones, Dean P. / Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells. In: Investigative Ophthalmology and Visual Science. 1999 ; Vol. 40, No. 5. pp. 959-966.
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N2 - PURPOSE. To determine the mechanism of oxidant-induced cell death in cultured human retinal pigment epithelium (hRPE). METHODS. Cultured hRPE cells were treated with different concentrations of a chemical oxidant, t- butylhydroperoxide (tBH), for different periods of time. Apoptosis was determined with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and flow cytometry. Mitochondrial membrane potential (mtΔΨ) was measured by rhodamine 123 staining and subsequent flow cytometry. Release of mitochondrial cytochrome c (cyt c) and cleavage of procaspase 3 and caspase substrates were determined by western blot analysis. RESULTS. t-Butylhydroperoxide caused time- and dose-dependent activation of apoptosis in hRPE, indicated by characteristic morphologic changes; TUNEL- positive labeling; phosphatidylserine (PS) exposure; and procaspase 3, poly(ADP-ribose)polymerase, lamin, and tubulin cleavage. An early decrease of mtΔΨ was observed before caspase activation, together with the release of mitochondrial cyt c. CONCLUSIONS. Results indicate that tBH can induce apoptosis in hRPE, probably by triggering the mitochondrial permeability transition, which results in swelling and release of mitochondrial intermembrane proteins.

AB - PURPOSE. To determine the mechanism of oxidant-induced cell death in cultured human retinal pigment epithelium (hRPE). METHODS. Cultured hRPE cells were treated with different concentrations of a chemical oxidant, t- butylhydroperoxide (tBH), for different periods of time. Apoptosis was determined with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and flow cytometry. Mitochondrial membrane potential (mtΔΨ) was measured by rhodamine 123 staining and subsequent flow cytometry. Release of mitochondrial cytochrome c (cyt c) and cleavage of procaspase 3 and caspase substrates were determined by western blot analysis. RESULTS. t-Butylhydroperoxide caused time- and dose-dependent activation of apoptosis in hRPE, indicated by characteristic morphologic changes; TUNEL- positive labeling; phosphatidylserine (PS) exposure; and procaspase 3, poly(ADP-ribose)polymerase, lamin, and tubulin cleavage. An early decrease of mtΔΨ was observed before caspase activation, together with the release of mitochondrial cyt c. CONCLUSIONS. Results indicate that tBH can induce apoptosis in hRPE, probably by triggering the mitochondrial permeability transition, which results in swelling and release of mitochondrial intermembrane proteins.

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