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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.


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AChE activity in Y79 cells treated with glucose. Y79 cells were pre-treated for 16–24 h in starvation medium (1% FBS and 1 mg/ml of glucose), and then with 3.5 mg/ml or 6 mg/ml glucose for 1 h or 24 h, after which Karnovsky and Roots assay was performed. Positive Karnovsky and Roots staining is seen in (A) on the right side compared to the negative control at the left. Scale bar is 50 μm. (B) Three independent experiments at which about 20 fields of cells (from each experiment) were counted for each treatment, calculated, and plotted as fold of control (cells cultured in starvation media containing 1% FBS and 1 mg/ml glucose) in histograms. Values are mean ± SEM, (N = 3). *p < 0.05.
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Figure 4: AChE activity in Y79 cells treated with glucose. Y79 cells were pre-treated for 16–24 h in starvation medium (1% FBS and 1 mg/ml of glucose), and then with 3.5 mg/ml or 6 mg/ml glucose for 1 h or 24 h, after which Karnovsky and Roots assay was performed. Positive Karnovsky and Roots staining is seen in (A) on the right side compared to the negative control at the left. Scale bar is 50 μm. (B) Three independent experiments at which about 20 fields of cells (from each experiment) were counted for each treatment, calculated, and plotted as fold of control (cells cultured in starvation media containing 1% FBS and 1 mg/ml glucose) in histograms. Values are mean ± SEM, (N = 3). *p < 0.05.

Mentions: To test whether the hyperglycemia-induced expression of AChE is also accompanied by increased AChE activity, we tested AChE activity in Y79 cells exposed to glucose concentrations that induced apoptosis. AChE activity, as demonstrated by Karnovsky and Roots staining (Karnovsky and Roots, 1964) (Figure 4A), was calculated relative to control conditions (1 mg/ml glucose), and was found to increase by 7.0 ± 1.9-fold in cells treated with 3.5 mg/ml glucose for 1 h. In agreement with AChE expression pattern, AChE activity decreased with time after exposure to high glucose (3.5 mg/ml) and after 24 h was only 1.3 ± 0.34-fold relative to control (Figure 4B). This decrease within 24 h was statistically significant (p < 0.05). AChE activity in cells exposed to 3.5 mg/ml glucose for 1 h was significantly (p < 0.05) higher compared to cells exposed for the same time period to 6 mg/ml glucose. AChE activity was not increased in cells treated with 6 mg/ml glucose for the same time intervals (Figure 4B).


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)

AChE activity in Y79 cells treated with glucose. Y79 cells were pre-treated for 16–24 h in starvation medium (1% FBS and 1 mg/ml of glucose), and then with 3.5 mg/ml or 6 mg/ml glucose for 1 h or 24 h, after which Karnovsky and Roots assay was performed. Positive Karnovsky and Roots staining is seen in (A) on the right side compared to the negative control at the left. Scale bar is 50 μm. (B) Three independent experiments at which about 20 fields of cells (from each experiment) were counted for each treatment, calculated, and plotted as fold of control (cells cultured in starvation media containing 1% FBS and 1 mg/ml glucose) in histograms. Values are mean ± SEM, (N = 3). *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368359&req=5

Figure 4: AChE activity in Y79 cells treated with glucose. Y79 cells were pre-treated for 16–24 h in starvation medium (1% FBS and 1 mg/ml of glucose), and then with 3.5 mg/ml or 6 mg/ml glucose for 1 h or 24 h, after which Karnovsky and Roots assay was performed. Positive Karnovsky and Roots staining is seen in (A) on the right side compared to the negative control at the left. Scale bar is 50 μm. (B) Three independent experiments at which about 20 fields of cells (from each experiment) were counted for each treatment, calculated, and plotted as fold of control (cells cultured in starvation media containing 1% FBS and 1 mg/ml glucose) in histograms. Values are mean ± SEM, (N = 3). *p < 0.05.
Mentions: To test whether the hyperglycemia-induced expression of AChE is also accompanied by increased AChE activity, we tested AChE activity in Y79 cells exposed to glucose concentrations that induced apoptosis. AChE activity, as demonstrated by Karnovsky and Roots staining (Karnovsky and Roots, 1964) (Figure 4A), was calculated relative to control conditions (1 mg/ml glucose), and was found to increase by 7.0 ± 1.9-fold in cells treated with 3.5 mg/ml glucose for 1 h. In agreement with AChE expression pattern, AChE activity decreased with time after exposure to high glucose (3.5 mg/ml) and after 24 h was only 1.3 ± 0.34-fold relative to control (Figure 4B). This decrease within 24 h was statistically significant (p < 0.05). AChE activity in cells exposed to 3.5 mg/ml glucose for 1 h was significantly (p < 0.05) higher compared to cells exposed for the same time period to 6 mg/ml glucose. AChE activity was not increased in cells treated with 6 mg/ml glucose for the same time intervals (Figure 4B).

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