Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways

Irma Charles, Abdelnaby Khalyfa, D. Maneesh Kumar, Raghu Krishnamoorthy, Rouel S. Roque, Nigel Cooper, Neeraj Agarwal

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Abstract

PURPOSE. Apoptosis-related signaling pathways were investigated in a cultured rat retinal ganglion cell (RGC-5) line deprived of growth factors after serum withdrawal from the culture medium. METHODS. RGC-5 cells were subjected to serum deprivation for 2 to 6 days and compared with RGC-5 cells cultured in growth medium containing 10% fetal calf serum. Cell viability was determined by a neutral red dye uptake assay. Apoptosis of RGC-5 cells was established by DNA laddering. The expression of various apoptosis-related genes was investigated by immunoblot analysis, and or reverse transcription polymerase chain reaction (RT-PCR) analysis. The redox state of the cell was determined by biochemical methods, including NF-κB binding activity by electrophoretic mobility gel shift assays (EMSA) and mitochondrial damage by JC-1 (5,5′, 6,6′-tetrachloro 1,1′,3,3′-tetraethylbenzimidazolyl- carbocyanine iodide) staining, using live cell confocal microscopy and cytosolic release of cytochrome c. RESULTS. Fifty percent cell loss was evident after 2 days of serum deprivation, as demonstrated by neutral red dye uptake assay. This cell loss was due to apoptotic cell death, as established by DNA laddering. The oxidative state of serum-deprived RGC-5 cells was perturbed as suggested by the increase in malonyldialdehyde (MDA) and a decrease in reduced glutathione (GSH) levels in cell lysates. The apoptosis of the RGC-5 cells was associated with the activation of caspase-3, -8, and -9, and increased levels of Bax with corresponding decreases in Bcl-2 levels and NF-κB (NF-κB) binding activity. Serum deprivation was also associated with a loss of mitochondrial function, as revealed by cytosolic release of cytochrome c and JC-1 staining of mitochondria of dying RGC-5 cells. CONCLUSIONS. Taken together, these results indicate that serum withdrawal induces apoptotic cell death in RGC-5 cells via mitochondrial pathways. These studies lead to the speculation that growth factor deprivation arising from blockade of retrograde transport of neurotrophins may involve similar mechanism(s) of retinal ganglion cell death in glaucoma.

Original languageEnglish
Pages (from-to)1330-1338
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume46
Issue number4
DOIs
StatePublished - 1 Apr 2005

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Retinal Ganglion Cells
Cell Death
Serum
Apoptosis
Neutral Red
Intercellular Signaling Peptides and Proteins
Coloring Agents
Carbocyanines
Cytochromes c1
Staining and Labeling
Caspase 8
DNA
Nerve Growth Factors
Iodides
Electrophoretic Mobility Shift Assay
Cytochromes c
Malondialdehyde
Confocal Microscopy
Caspase 3
Glaucoma

Cite this

Charles, Irma ; Khalyfa, Abdelnaby ; Kumar, D. Maneesh ; Krishnamoorthy, Raghu ; Roque, Rouel S. ; Cooper, Nigel ; Agarwal, Neeraj. / Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways. In: Investigative Ophthalmology and Visual Science. 2005 ; Vol. 46, No. 4. pp. 1330-1338.
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title = "Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways",
abstract = "PURPOSE. Apoptosis-related signaling pathways were investigated in a cultured rat retinal ganglion cell (RGC-5) line deprived of growth factors after serum withdrawal from the culture medium. METHODS. RGC-5 cells were subjected to serum deprivation for 2 to 6 days and compared with RGC-5 cells cultured in growth medium containing 10{\%} fetal calf serum. Cell viability was determined by a neutral red dye uptake assay. Apoptosis of RGC-5 cells was established by DNA laddering. The expression of various apoptosis-related genes was investigated by immunoblot analysis, and or reverse transcription polymerase chain reaction (RT-PCR) analysis. The redox state of the cell was determined by biochemical methods, including NF-κB binding activity by electrophoretic mobility gel shift assays (EMSA) and mitochondrial damage by JC-1 (5,5′, 6,6′-tetrachloro 1,1′,3,3′-tetraethylbenzimidazolyl- carbocyanine iodide) staining, using live cell confocal microscopy and cytosolic release of cytochrome c. RESULTS. Fifty percent cell loss was evident after 2 days of serum deprivation, as demonstrated by neutral red dye uptake assay. This cell loss was due to apoptotic cell death, as established by DNA laddering. The oxidative state of serum-deprived RGC-5 cells was perturbed as suggested by the increase in malonyldialdehyde (MDA) and a decrease in reduced glutathione (GSH) levels in cell lysates. The apoptosis of the RGC-5 cells was associated with the activation of caspase-3, -8, and -9, and increased levels of Bax with corresponding decreases in Bcl-2 levels and NF-κB (NF-κB) binding activity. Serum deprivation was also associated with a loss of mitochondrial function, as revealed by cytosolic release of cytochrome c and JC-1 staining of mitochondria of dying RGC-5 cells. CONCLUSIONS. Taken together, these results indicate that serum withdrawal induces apoptotic cell death in RGC-5 cells via mitochondrial pathways. These studies lead to the speculation that growth factor deprivation arising from blockade of retrograde transport of neurotrophins may involve similar mechanism(s) of retinal ganglion cell death in glaucoma.",
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Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways. / Charles, Irma; Khalyfa, Abdelnaby; Kumar, D. Maneesh; Krishnamoorthy, Raghu; Roque, Rouel S.; Cooper, Nigel; Agarwal, Neeraj.

In: Investigative Ophthalmology and Visual Science, Vol. 46, No. 4, 01.04.2005, p. 1330-1338.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways

AU - Charles, Irma

AU - Khalyfa, Abdelnaby

AU - Kumar, D. Maneesh

AU - Krishnamoorthy, Raghu

AU - Roque, Rouel S.

AU - Cooper, Nigel

AU - Agarwal, Neeraj

PY - 2005/4/1

Y1 - 2005/4/1

N2 - PURPOSE. Apoptosis-related signaling pathways were investigated in a cultured rat retinal ganglion cell (RGC-5) line deprived of growth factors after serum withdrawal from the culture medium. METHODS. RGC-5 cells were subjected to serum deprivation for 2 to 6 days and compared with RGC-5 cells cultured in growth medium containing 10% fetal calf serum. Cell viability was determined by a neutral red dye uptake assay. Apoptosis of RGC-5 cells was established by DNA laddering. The expression of various apoptosis-related genes was investigated by immunoblot analysis, and or reverse transcription polymerase chain reaction (RT-PCR) analysis. The redox state of the cell was determined by biochemical methods, including NF-κB binding activity by electrophoretic mobility gel shift assays (EMSA) and mitochondrial damage by JC-1 (5,5′, 6,6′-tetrachloro 1,1′,3,3′-tetraethylbenzimidazolyl- carbocyanine iodide) staining, using live cell confocal microscopy and cytosolic release of cytochrome c. RESULTS. Fifty percent cell loss was evident after 2 days of serum deprivation, as demonstrated by neutral red dye uptake assay. This cell loss was due to apoptotic cell death, as established by DNA laddering. The oxidative state of serum-deprived RGC-5 cells was perturbed as suggested by the increase in malonyldialdehyde (MDA) and a decrease in reduced glutathione (GSH) levels in cell lysates. The apoptosis of the RGC-5 cells was associated with the activation of caspase-3, -8, and -9, and increased levels of Bax with corresponding decreases in Bcl-2 levels and NF-κB (NF-κB) binding activity. Serum deprivation was also associated with a loss of mitochondrial function, as revealed by cytosolic release of cytochrome c and JC-1 staining of mitochondria of dying RGC-5 cells. CONCLUSIONS. Taken together, these results indicate that serum withdrawal induces apoptotic cell death in RGC-5 cells via mitochondrial pathways. These studies lead to the speculation that growth factor deprivation arising from blockade of retrograde transport of neurotrophins may involve similar mechanism(s) of retinal ganglion cell death in glaucoma.

AB - PURPOSE. Apoptosis-related signaling pathways were investigated in a cultured rat retinal ganglion cell (RGC-5) line deprived of growth factors after serum withdrawal from the culture medium. METHODS. RGC-5 cells were subjected to serum deprivation for 2 to 6 days and compared with RGC-5 cells cultured in growth medium containing 10% fetal calf serum. Cell viability was determined by a neutral red dye uptake assay. Apoptosis of RGC-5 cells was established by DNA laddering. The expression of various apoptosis-related genes was investigated by immunoblot analysis, and or reverse transcription polymerase chain reaction (RT-PCR) analysis. The redox state of the cell was determined by biochemical methods, including NF-κB binding activity by electrophoretic mobility gel shift assays (EMSA) and mitochondrial damage by JC-1 (5,5′, 6,6′-tetrachloro 1,1′,3,3′-tetraethylbenzimidazolyl- carbocyanine iodide) staining, using live cell confocal microscopy and cytosolic release of cytochrome c. RESULTS. Fifty percent cell loss was evident after 2 days of serum deprivation, as demonstrated by neutral red dye uptake assay. This cell loss was due to apoptotic cell death, as established by DNA laddering. The oxidative state of serum-deprived RGC-5 cells was perturbed as suggested by the increase in malonyldialdehyde (MDA) and a decrease in reduced glutathione (GSH) levels in cell lysates. The apoptosis of the RGC-5 cells was associated with the activation of caspase-3, -8, and -9, and increased levels of Bax with corresponding decreases in Bcl-2 levels and NF-κB (NF-κB) binding activity. Serum deprivation was also associated with a loss of mitochondrial function, as revealed by cytosolic release of cytochrome c and JC-1 staining of mitochondria of dying RGC-5 cells. CONCLUSIONS. Taken together, these results indicate that serum withdrawal induces apoptotic cell death in RGC-5 cells via mitochondrial pathways. These studies lead to the speculation that growth factor deprivation arising from blockade of retrograde transport of neurotrophins may involve similar mechanism(s) of retinal ganglion cell death in glaucoma.

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U2 - 10.1167/iovs.04-0363

DO - 10.1167/iovs.04-0363

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JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

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