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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

Cleavage of PARP in nuclear extracts of sh-RNA lenti-infected Y79 cells treated by adding glucose. Nuclear extracts were prepared from starved Y79 sh-RNA lenti-infected and selected cells, after raising glucose concentration to 3.5 mg/ml glucose for 1 h or keeping the cells in control (1 mg/ml glucose) conditions (–). Extracts were run on SDS-PAGE, blotted and probed with anti- PARP as described under Materials and Methods. Five independent western experiments were conducted; one is shown in (A). Densitometry values were calculated for cleaved to non-cleaved ratios, and then calculated as fold of cells incubated under starvation medium (containing 1% FBS and 1 mg/ml glucose) and plotted in histograms as shown in (B). Values are mean ± SEM, (N = 5). *p = 0.05.
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Figure 7: Cleavage of PARP in nuclear extracts of sh-RNA lenti-infected Y79 cells treated by adding glucose. Nuclear extracts were prepared from starved Y79 sh-RNA lenti-infected and selected cells, after raising glucose concentration to 3.5 mg/ml glucose for 1 h or keeping the cells in control (1 mg/ml glucose) conditions (–). Extracts were run on SDS-PAGE, blotted and probed with anti- PARP as described under Materials and Methods. Five independent western experiments were conducted; one is shown in (A). Densitometry values were calculated for cleaved to non-cleaved ratios, and then calculated as fold of cells incubated under starvation medium (containing 1% FBS and 1 mg/ml glucose) and plotted in histograms as shown in (B). Values are mean ± SEM, (N = 5). *p = 0.05.

Mentions: The TUNEL results were strengthened by measurements of PARP cleavage in nuclear extracts of the sh-Lenti infected lines; sh-control, sh-4, and sh-5. The western blot shown in Figure 7A and the densitometry results plotted in Figure 7B, show that PARP was cleaved by almost 3-fold in sh-control Lenti infected Y79 cells following 1 h incubation in 3.5 mg/ml glucose compared to cells remaining in 1 mg/ml glucose. In contrast, cleavage of PARP could not be detected in the sh-4 and sh-5 Lenti-infected Y79 cells, where AChE translation was blocked. The TUNEL and PARP results and the AChE protein expression data seen in Figures 6 and 7, suggest that inhibition of AChE protein expression prevents Y79 cells from entering into the apoptotic cascade.


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)

Cleavage of PARP in nuclear extracts of sh-RNA lenti-infected Y79 cells treated by adding glucose. Nuclear extracts were prepared from starved Y79 sh-RNA lenti-infected and selected cells, after raising glucose concentration to 3.5 mg/ml glucose for 1 h or keeping the cells in control (1 mg/ml glucose) conditions (–). Extracts were run on SDS-PAGE, blotted and probed with anti- PARP as described under Materials and Methods. Five independent western experiments were conducted; one is shown in (A). Densitometry values were calculated for cleaved to non-cleaved ratios, and then calculated as fold of cells incubated under starvation medium (containing 1% FBS and 1 mg/ml glucose) and plotted in histograms as shown in (B). Values are mean ± SEM, (N = 5). *p = 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Cleavage of PARP in nuclear extracts of sh-RNA lenti-infected Y79 cells treated by adding glucose. Nuclear extracts were prepared from starved Y79 sh-RNA lenti-infected and selected cells, after raising glucose concentration to 3.5 mg/ml glucose for 1 h or keeping the cells in control (1 mg/ml glucose) conditions (–). Extracts were run on SDS-PAGE, blotted and probed with anti- PARP as described under Materials and Methods. Five independent western experiments were conducted; one is shown in (A). Densitometry values were calculated for cleaved to non-cleaved ratios, and then calculated as fold of cells incubated under starvation medium (containing 1% FBS and 1 mg/ml glucose) and plotted in histograms as shown in (B). Values are mean ± SEM, (N = 5). *p = 0.05.
Mentions: The TUNEL results were strengthened by measurements of PARP cleavage in nuclear extracts of the sh-Lenti infected lines; sh-control, sh-4, and sh-5. The western blot shown in Figure 7A and the densitometry results plotted in Figure 7B, show that PARP was cleaved by almost 3-fold in sh-control Lenti infected Y79 cells following 1 h incubation in 3.5 mg/ml glucose compared to cells remaining in 1 mg/ml glucose. In contrast, cleavage of PARP could not be detected in the sh-4 and sh-5 Lenti-infected Y79 cells, where AChE translation was blocked. The TUNEL and PARP results and the AChE protein expression data seen in Figures 6 and 7, suggest that inhibition of AChE protein expression prevents Y79 cells from entering into the apoptotic cascade.

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