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Acetylcholinesterase (AChE) is an important link in the apoptotic pathway induced by hyperglycemia in Y79 retinoblastoma cell line.

Masha'our RS, Heinrich R, Garzozi HJ, Perlman I - Front Mol Neurosci (2012)

Bottom Line: Y79 retinoblastoma cells were incubated in starvation media (1% FBS and 1 mg/ml glucose) for 16-24 h, and then exposed to hyperglycemic environment by raising extracellular glucose concentrations to a final level of 3.5 mg/ml or 6 mg/ml.Apoptosis was detected by the cleavage of Poly ADP-ribose polymerase (PARP) using western blot, and by Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) assay.Treating Y79 cells with 3.5 mg/ml of glucose, but not with 3.5 mg/ml mannitol, induced apoptosis which was confirmed by TUNEL assay and by cleavage of PARP.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Bnai-Zion Medical Center Haifa, Israel.

ABSTRACT
Acetylcholinesterase (AChE) expression was found to be induced in the mammalian CNS, including the retina, by different types of stress leading to cellular apoptosis. Here, we tested possible involvement of AChE in hyperglycemia-induced apoptosis in a retinal cell line. Y79 retinoblastoma cells were incubated in starvation media (1% FBS and 1 mg/ml glucose) for 16-24 h, and then exposed to hyperglycemic environment by raising extracellular glucose concentrations to a final level of 3.5 mg/ml or 6 mg/ml. Similar levels of mannitol were used as control for hyperosmolarity. Cells were harvested at different time intervals for analysis of apoptosis and AChE protein expression. Apoptosis was detected by the cleavage of Poly ADP-ribose polymerase (PARP) using western blot, and by Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) assay. AChE protein expression and activity was detected by western blot and by the Karnovsky and Roots method, respectively. Mission(TM) shRNA for AChE was used to inhibit AChE protein expression. Treating Y79 cells with 3.5 mg/ml of glucose, but not with 3.5 mg/ml mannitol, induced apoptosis which was confirmed by TUNEL assay and by cleavage of PARP. A part of the signaling pathway accompanying the apoptotic process involved up-regulation of the AChE-R variant and an N-extended AChE variant as verified at the mRNA and protein level. Inhibition of AChE protein expression by shRNA protected Y79 cell from entering the apoptotic pathway. Our data suggest that expression of an N-extended AChE variant, most probably an R isoform, is involved in the apoptotic pathway caused by hyperglycemia in Y79 cells.

No MeSH data available.


Related in: MedlinePlus

Hyperglicemia (3.5 mg/ml or 6 mg/ml of glucose)-induced apoptosis induction in Y79 cells. (A) Cells tested by TUNEL to detect apoptosis (green) and stained with DAPI for detecting nuclei (blue). Y79 cells were pre-treated for 16–24 h in starvation medium (1% FBS and 1 mg/ml glucose), and then incubated for 1 h with 3.5 mg/ml glucose did not show signs of apoptosis (left); while cells incubated for 24 h in 3.5 mg/ml or 6 mg/ml glucose are TUNEL positive. Scale bar is 50 μm. (B) TUNEL positive cells were counted and calculated as fold of control (cells cultured in starvation media containing 1% FBS and 1 mg/ml glucose) and plotted in histograms. Values are means ± SEM, (N = 3). *p < 0.05, #p = 0.05.
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Figure 1: Hyperglicemia (3.5 mg/ml or 6 mg/ml of glucose)-induced apoptosis induction in Y79 cells. (A) Cells tested by TUNEL to detect apoptosis (green) and stained with DAPI for detecting nuclei (blue). Y79 cells were pre-treated for 16–24 h in starvation medium (1% FBS and 1 mg/ml glucose), and then incubated for 1 h with 3.5 mg/ml glucose did not show signs of apoptosis (left); while cells incubated for 24 h in 3.5 mg/ml or 6 mg/ml glucose are TUNEL positive. Scale bar is 50 μm. (B) TUNEL positive cells were counted and calculated as fold of control (cells cultured in starvation media containing 1% FBS and 1 mg/ml glucose) and plotted in histograms. Values are means ± SEM, (N = 3). *p < 0.05, #p = 0.05.

Mentions: Keeping Y79 cells for 1 or 24 h in 1 mg/ml glucose (5.5 mM), mimicking physiologic conditions did not induce apoptosis (Figure 1A). Raising the level of glucose to 3.5 mg/ml (19.4 mM) or to 6 mg/ml (33.3 mM), for 1 h also did not result in apoptosis (Figure 1A). However, after 24 h in high glucose (3.5 mg/ml or 6 mg/ml—mimicking hyperglycemia in diabetes), apoptosis was stimulated as evident by abundance of TUNEL positive cells (Figure 1A).


Acetylcholinesterase (AChE) is an important link in the apoptotic pathway induced by hyperglycemia in Y79 retinoblastoma cell line.

Masha'our RS, Heinrich R, Garzozi HJ, Perlman I - Front Mol Neurosci (2012)

Hyperglicemia (3.5 mg/ml or 6 mg/ml of glucose)-induced apoptosis induction in Y79 cells. (A) Cells tested by TUNEL to detect apoptosis (green) and stained with DAPI for detecting nuclei (blue). Y79 cells were pre-treated for 16–24 h in starvation medium (1% FBS and 1 mg/ml glucose), and then incubated for 1 h with 3.5 mg/ml glucose did not show signs of apoptosis (left); while cells incubated for 24 h in 3.5 mg/ml or 6 mg/ml glucose are TUNEL positive. Scale bar is 50 μm. (B) TUNEL positive cells were counted and calculated as fold of control (cells cultured in starvation media containing 1% FBS and 1 mg/ml glucose) and plotted in histograms. Values are means ± SEM, (N = 3). *p < 0.05, #p = 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368359&req=5

Figure 1: Hyperglicemia (3.5 mg/ml or 6 mg/ml of glucose)-induced apoptosis induction in Y79 cells. (A) Cells tested by TUNEL to detect apoptosis (green) and stained with DAPI for detecting nuclei (blue). Y79 cells were pre-treated for 16–24 h in starvation medium (1% FBS and 1 mg/ml glucose), and then incubated for 1 h with 3.5 mg/ml glucose did not show signs of apoptosis (left); while cells incubated for 24 h in 3.5 mg/ml or 6 mg/ml glucose are TUNEL positive. Scale bar is 50 μm. (B) TUNEL positive cells were counted and calculated as fold of control (cells cultured in starvation media containing 1% FBS and 1 mg/ml glucose) and plotted in histograms. Values are means ± SEM, (N = 3). *p < 0.05, #p = 0.05.
Mentions: Keeping Y79 cells for 1 or 24 h in 1 mg/ml glucose (5.5 mM), mimicking physiologic conditions did not induce apoptosis (Figure 1A). Raising the level of glucose to 3.5 mg/ml (19.4 mM) or to 6 mg/ml (33.3 mM), for 1 h also did not result in apoptosis (Figure 1A). However, after 24 h in high glucose (3.5 mg/ml or 6 mg/ml—mimicking hyperglycemia in diabetes), apoptosis was stimulated as evident by abundance of TUNEL positive cells (Figure 1A).

Bottom Line: Y79 retinoblastoma cells were incubated in starvation media (1% FBS and 1 mg/ml glucose) for 16-24 h, and then exposed to hyperglycemic environment by raising extracellular glucose concentrations to a final level of 3.5 mg/ml or 6 mg/ml.Apoptosis was detected by the cleavage of Poly ADP-ribose polymerase (PARP) using western blot, and by Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) assay.Treating Y79 cells with 3.5 mg/ml of glucose, but not with 3.5 mg/ml mannitol, induced apoptosis which was confirmed by TUNEL assay and by cleavage of PARP.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Bnai-Zion Medical Center Haifa, Israel.

ABSTRACT
Acetylcholinesterase (AChE) expression was found to be induced in the mammalian CNS, including the retina, by different types of stress leading to cellular apoptosis. Here, we tested possible involvement of AChE in hyperglycemia-induced apoptosis in a retinal cell line. Y79 retinoblastoma cells were incubated in starvation media (1% FBS and 1 mg/ml glucose) for 16-24 h, and then exposed to hyperglycemic environment by raising extracellular glucose concentrations to a final level of 3.5 mg/ml or 6 mg/ml. Similar levels of mannitol were used as control for hyperosmolarity. Cells were harvested at different time intervals for analysis of apoptosis and AChE protein expression. Apoptosis was detected by the cleavage of Poly ADP-ribose polymerase (PARP) using western blot, and by Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) assay. AChE protein expression and activity was detected by western blot and by the Karnovsky and Roots method, respectively. Mission(TM) shRNA for AChE was used to inhibit AChE protein expression. Treating Y79 cells with 3.5 mg/ml of glucose, but not with 3.5 mg/ml mannitol, induced apoptosis which was confirmed by TUNEL assay and by cleavage of PARP. A part of the signaling pathway accompanying the apoptotic process involved up-regulation of the AChE-R variant and an N-extended AChE variant as verified at the mRNA and protein level. Inhibition of AChE protein expression by shRNA protected Y79 cell from entering the apoptotic pathway. Our data suggest that expression of an N-extended AChE variant, most probably an R isoform, is involved in the apoptotic pathway caused by hyperglycemia in Y79 cells.

No MeSH data available.


Related in: MedlinePlus