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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

AChE expression in Y79 cells treated by adding glucose or mannitol. Y79 cells were treated first for 16–24 h in starvation medium (1% FBS and 1 mg/ml glucose), and then incubated with 3.5 mg/ml or 6 mg/ml of glucose or mannitol for 1 or 2 h. Following 1 or 2 h incubation cytoplasmic extracts were prepared and run on SDS-PAGE, blotted, and probed with anti- AChE or anti-α-Tubulin (for loading control) as described under Materials and Methods. The western blot shown is one of two independent experiments.
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Figure 3: AChE expression in Y79 cells treated by adding glucose or mannitol. Y79 cells were treated first for 16–24 h in starvation medium (1% FBS and 1 mg/ml glucose), and then incubated with 3.5 mg/ml or 6 mg/ml of glucose or mannitol for 1 or 2 h. Following 1 or 2 h incubation cytoplasmic extracts were prepared and run on SDS-PAGE, blotted, and probed with anti- AChE or anti-α-Tubulin (for loading control) as described under Materials and Methods. The western blot shown is one of two independent experiments.

Mentions: Figure 3 shows western blot analysis for AChE expression in Y79 cells following 1 h or 2 h incubation in different media. AChE expression was increased in Y79 cells treated with 3.5 mg/ml glucose (G) for 1 h and decreased after 2 h to control levels. AChE expression was not increased in cells treated with 6 mg/ml glucose or in cells treated with either 3.5 mg/ml or 6 mg/ml mannitol (M). The dispersed bands seen in Figure 3 in the range of 50–75 kDa probably reflect the formation of covalent PRiMA-AChE complexes or differences in the degree of glycosylation of AChE subunits (Darreh-Shori et al., 2004).


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 expression in Y79 cells treated by adding glucose or mannitol. Y79 cells were treated first for 16–24 h in starvation medium (1% FBS and 1 mg/ml glucose), and then incubated with 3.5 mg/ml or 6 mg/ml of glucose or mannitol for 1 or 2 h. Following 1 or 2 h incubation cytoplasmic extracts were prepared and run on SDS-PAGE, blotted, and probed with anti- AChE or anti-α-Tubulin (for loading control) as described under Materials and Methods. The western blot shown is one of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: AChE expression in Y79 cells treated by adding glucose or mannitol. Y79 cells were treated first for 16–24 h in starvation medium (1% FBS and 1 mg/ml glucose), and then incubated with 3.5 mg/ml or 6 mg/ml of glucose or mannitol for 1 or 2 h. Following 1 or 2 h incubation cytoplasmic extracts were prepared and run on SDS-PAGE, blotted, and probed with anti- AChE or anti-α-Tubulin (for loading control) as described under Materials and Methods. The western blot shown is one of two independent experiments.
Mentions: Figure 3 shows western blot analysis for AChE expression in Y79 cells following 1 h or 2 h incubation in different media. AChE expression was increased in Y79 cells treated with 3.5 mg/ml glucose (G) for 1 h and decreased after 2 h to control levels. AChE expression was not increased in cells treated with 6 mg/ml glucose or in cells treated with either 3.5 mg/ml or 6 mg/ml mannitol (M). The dispersed bands seen in Figure 3 in the range of 50–75 kDa probably reflect the formation of covalent PRiMA-AChE complexes or differences in the degree of glycosylation of AChE subunits (Darreh-Shori et al., 2004).

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