Sensitivity of staurosporine-induced differentiated RGC-5 cells to homocysteine sensitivity of RGC-5 cells to homocysteine

Preethi S. Ganapathy, Ying Dun, Yonju Ha, Jennifer Duplantier, John Bradley Allen, Amina Farooq, B. Renee Bozard, Sylvia B. Smith

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Purpose:Homocysteine is implicated in ganglion cell death associated with glaucoma. To understand mechanisms of homocysteine-induced cell death, we analyzed the sensitivity of the RGC-5 cell line, differentiated using staurosporine, to physiologically-relevant levels of the excitotoxic amino acid homocysteine. Methods:RGC-5 cells were differentiated 24hr using 316nM staurosporine and tested for expression of Thy 1.2 via immunodetection, RT-PCR, and immunoblotting. The sensitivity of staurosporine-differentiated RGC-5 cells to physiological levels of homocysteine (50, 100, 250 μM) and to high levels of homocysteine (1mM), glutamate (1mM), and oxidative stress (25 μM:10 mU/ml xanthine:xanthine oxidase) was assessed by TUNEL assay and by immunodetection of cleaved caspase-3. The sensitivity of undifferentiated RGC-5 cells to high (1, 5, and 10mM) homocysteine was also examined. Results:Undifferentiated RGC-5 cells express Thy 1.2 mRNA and protein. Staurosporine-differentiated RGC-5 cells extend neurite processes and express Thy 1.2 after 24hr differentiation; they express NF-L after 1 and 3 days differentiation. Treatment of staurosporine -differentiated RGC-5 cells with 50, 100, or 250 M homocysteine did not alter neurite processes nor induce cell death (detected by TUNEL and active caspase-3) to a level greater than that observed in the control (non-homocysteine-treated, staurosporine-differentiated) cells. The 1mM dosage of homocysteine in staurosporine-differentiated RGC-5 cells also did not induce cell death above control levels, although 18hr treatment of non-differentiated RGC-5 cells with 5mM homocysteine decreased survival by 50%. Conclusions:RGC-5 cells differentiated for 24hr with 316nM staurosporine project robust neurite processes and are positive for ganglion cell markers consistent with a more neuronal phenotype than non-staurosporine-differentiated RGC-5 cells. However, concentrations of homocysteine known to induce ganglion cell death in vivo and in primary ganglion cells are not sufficient to induce death of RGC-5 cells, even when they are differentiated with staurosporine.

Original languageEnglish (US)
Pages (from-to)80-90
Number of pages11
JournalCurrent Eye Research
Volume35
Issue number1
DOIs
StatePublished - Jan 2010
Externally publishedYes

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Staurosporine
Homocysteine
Cell Death
Ganglia
Neurites
In Situ Nick-End Labeling
Caspase 3
Xanthine
Xanthine Oxidase
Immunoblotting
Glaucoma
Glutamic Acid

Keywords

  • Apoptosis
  • Excitotoxicity
  • Hyperhomocysteinemia
  • Mouse retinal cell line
  • Neuronal differentiation
  • Retinal ganglion cells

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Sensitivity of staurosporine-induced differentiated RGC-5 cells to homocysteine sensitivity of RGC-5 cells to homocysteine. / Ganapathy, Preethi S.; Dun, Ying; Ha, Yonju; Duplantier, Jennifer; Allen, John Bradley; Farooq, Amina; Bozard, B. Renee; Smith, Sylvia B.

In: Current Eye Research, Vol. 35, No. 1, 01.2010, p. 80-90.

Research output: Contribution to journalArticle

Ganapathy, Preethi S. ; Dun, Ying ; Ha, Yonju ; Duplantier, Jennifer ; Allen, John Bradley ; Farooq, Amina ; Bozard, B. Renee ; Smith, Sylvia B. / Sensitivity of staurosporine-induced differentiated RGC-5 cells to homocysteine sensitivity of RGC-5 cells to homocysteine. In: Current Eye Research. 2010 ; Vol. 35, No. 1. pp. 80-90.
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abstract = "Purpose:Homocysteine is implicated in ganglion cell death associated with glaucoma. To understand mechanisms of homocysteine-induced cell death, we analyzed the sensitivity of the RGC-5 cell line, differentiated using staurosporine, to physiologically-relevant levels of the excitotoxic amino acid homocysteine. Methods:RGC-5 cells were differentiated 24hr using 316nM staurosporine and tested for expression of Thy 1.2 via immunodetection, RT-PCR, and immunoblotting. The sensitivity of staurosporine-differentiated RGC-5 cells to physiological levels of homocysteine (50, 100, 250 μM) and to high levels of homocysteine (1mM), glutamate (1mM), and oxidative stress (25 μM:10 mU/ml xanthine:xanthine oxidase) was assessed by TUNEL assay and by immunodetection of cleaved caspase-3. The sensitivity of undifferentiated RGC-5 cells to high (1, 5, and 10mM) homocysteine was also examined. Results:Undifferentiated RGC-5 cells express Thy 1.2 mRNA and protein. Staurosporine-differentiated RGC-5 cells extend neurite processes and express Thy 1.2 after 24hr differentiation; they express NF-L after 1 and 3 days differentiation. Treatment of staurosporine -differentiated RGC-5 cells with 50, 100, or 250 M homocysteine did not alter neurite processes nor induce cell death (detected by TUNEL and active caspase-3) to a level greater than that observed in the control (non-homocysteine-treated, staurosporine-differentiated) cells. The 1mM dosage of homocysteine in staurosporine-differentiated RGC-5 cells also did not induce cell death above control levels, although 18hr treatment of non-differentiated RGC-5 cells with 5mM homocysteine decreased survival by 50{\%}. Conclusions:RGC-5 cells differentiated for 24hr with 316nM staurosporine project robust neurite processes and are positive for ganglion cell markers consistent with a more neuronal phenotype than non-staurosporine-differentiated RGC-5 cells. However, concentrations of homocysteine known to induce ganglion cell death in vivo and in primary ganglion cells are not sufficient to induce death of RGC-5 cells, even when they are differentiated with staurosporine.",
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T1 - Sensitivity of staurosporine-induced differentiated RGC-5 cells to homocysteine sensitivity of RGC-5 cells to homocysteine

AU - Ganapathy, Preethi S.

AU - Dun, Ying

AU - Ha, Yonju

AU - Duplantier, Jennifer

AU - Allen, John Bradley

AU - Farooq, Amina

AU - Bozard, B. Renee

AU - Smith, Sylvia B.

PY - 2010/1

Y1 - 2010/1

N2 - Purpose:Homocysteine is implicated in ganglion cell death associated with glaucoma. To understand mechanisms of homocysteine-induced cell death, we analyzed the sensitivity of the RGC-5 cell line, differentiated using staurosporine, to physiologically-relevant levels of the excitotoxic amino acid homocysteine. Methods:RGC-5 cells were differentiated 24hr using 316nM staurosporine and tested for expression of Thy 1.2 via immunodetection, RT-PCR, and immunoblotting. The sensitivity of staurosporine-differentiated RGC-5 cells to physiological levels of homocysteine (50, 100, 250 μM) and to high levels of homocysteine (1mM), glutamate (1mM), and oxidative stress (25 μM:10 mU/ml xanthine:xanthine oxidase) was assessed by TUNEL assay and by immunodetection of cleaved caspase-3. The sensitivity of undifferentiated RGC-5 cells to high (1, 5, and 10mM) homocysteine was also examined. Results:Undifferentiated RGC-5 cells express Thy 1.2 mRNA and protein. Staurosporine-differentiated RGC-5 cells extend neurite processes and express Thy 1.2 after 24hr differentiation; they express NF-L after 1 and 3 days differentiation. Treatment of staurosporine -differentiated RGC-5 cells with 50, 100, or 250 M homocysteine did not alter neurite processes nor induce cell death (detected by TUNEL and active caspase-3) to a level greater than that observed in the control (non-homocysteine-treated, staurosporine-differentiated) cells. The 1mM dosage of homocysteine in staurosporine-differentiated RGC-5 cells also did not induce cell death above control levels, although 18hr treatment of non-differentiated RGC-5 cells with 5mM homocysteine decreased survival by 50%. Conclusions:RGC-5 cells differentiated for 24hr with 316nM staurosporine project robust neurite processes and are positive for ganglion cell markers consistent with a more neuronal phenotype than non-staurosporine-differentiated RGC-5 cells. However, concentrations of homocysteine known to induce ganglion cell death in vivo and in primary ganglion cells are not sufficient to induce death of RGC-5 cells, even when they are differentiated with staurosporine.

AB - Purpose:Homocysteine is implicated in ganglion cell death associated with glaucoma. To understand mechanisms of homocysteine-induced cell death, we analyzed the sensitivity of the RGC-5 cell line, differentiated using staurosporine, to physiologically-relevant levels of the excitotoxic amino acid homocysteine. Methods:RGC-5 cells were differentiated 24hr using 316nM staurosporine and tested for expression of Thy 1.2 via immunodetection, RT-PCR, and immunoblotting. The sensitivity of staurosporine-differentiated RGC-5 cells to physiological levels of homocysteine (50, 100, 250 μM) and to high levels of homocysteine (1mM), glutamate (1mM), and oxidative stress (25 μM:10 mU/ml xanthine:xanthine oxidase) was assessed by TUNEL assay and by immunodetection of cleaved caspase-3. The sensitivity of undifferentiated RGC-5 cells to high (1, 5, and 10mM) homocysteine was also examined. Results:Undifferentiated RGC-5 cells express Thy 1.2 mRNA and protein. Staurosporine-differentiated RGC-5 cells extend neurite processes and express Thy 1.2 after 24hr differentiation; they express NF-L after 1 and 3 days differentiation. Treatment of staurosporine -differentiated RGC-5 cells with 50, 100, or 250 M homocysteine did not alter neurite processes nor induce cell death (detected by TUNEL and active caspase-3) to a level greater than that observed in the control (non-homocysteine-treated, staurosporine-differentiated) cells. The 1mM dosage of homocysteine in staurosporine-differentiated RGC-5 cells also did not induce cell death above control levels, although 18hr treatment of non-differentiated RGC-5 cells with 5mM homocysteine decreased survival by 50%. Conclusions:RGC-5 cells differentiated for 24hr with 316nM staurosporine project robust neurite processes and are positive for ganglion cell markers consistent with a more neuronal phenotype than non-staurosporine-differentiated RGC-5 cells. However, concentrations of homocysteine known to induce ganglion cell death in vivo and in primary ganglion cells are not sufficient to induce death of RGC-5 cells, even when they are differentiated with staurosporine.

KW - Apoptosis

KW - Excitotoxicity

KW - Hyperhomocysteinemia

KW - Mouse retinal cell line

KW - Neuronal differentiation

KW - Retinal ganglion cells

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