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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

mRNA expression of N-extended AChE, AChE-S, and AChE-R isoforms following treatment of Y79 cells with glucose for 1 h. 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 glucose for different time intervals. Total RNA was extracted and cDNA was prepared for real-time PCR procedure as described under Materials and Methods. Results are presented as fold of control cells cultured in starvation media. Values are means ± SEM, (N = 4). *p < 0.05, #p = 0.12.
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Figure 5: mRNA expression of N-extended AChE, AChE-S, and AChE-R isoforms following treatment of Y79 cells with glucose for 1 h. 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 glucose for different time intervals. Total RNA was extracted and cDNA was prepared for real-time PCR procedure as described under Materials and Methods. Results are presented as fold of control cells cultured in starvation media. Values are means ± SEM, (N = 4). *p < 0.05, #p = 0.12.

Mentions: Since AChE was detected in the cytoplasmic (Figure 3) fraction, and in part of the experiments also in the membranous fraction (data not shown), we hypothesized that the isoforms detected by the western blot assays might be the short cytoplasmic R monomeric soluble isoform, and the membranous one might be the longer N-extended AChE (R or S variants) and/or the AChE-S variant. To test this hypothesis, Reverse Transcription (RT) real-time PCR was performed. As seen in Figure 5, mRNA expression levels corresponding to the AChE-S isoform were low and did not change significantly after 15-, 30- or 60- min incubation with 3.5 mg/ml of glucose. However, mRNA expression transcripts tested for the AChE-R or the N-extended isoforms, were significantly (p < 0.05) up-regulated by 1.5 ± 0.15 and by 1.4 ± 0.14-fold respectively, compared to control Y79 cells.


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)

mRNA expression of N-extended AChE, AChE-S, and AChE-R isoforms following treatment of Y79 cells with glucose for 1 h. 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 glucose for different time intervals. Total RNA was extracted and cDNA was prepared for real-time PCR procedure as described under Materials and Methods. Results are presented as fold of control cells cultured in starvation media. Values are means ± SEM, (N = 4). *p < 0.05, #p = 0.12.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: mRNA expression of N-extended AChE, AChE-S, and AChE-R isoforms following treatment of Y79 cells with glucose for 1 h. 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 glucose for different time intervals. Total RNA was extracted and cDNA was prepared for real-time PCR procedure as described under Materials and Methods. Results are presented as fold of control cells cultured in starvation media. Values are means ± SEM, (N = 4). *p < 0.05, #p = 0.12.
Mentions: Since AChE was detected in the cytoplasmic (Figure 3) fraction, and in part of the experiments also in the membranous fraction (data not shown), we hypothesized that the isoforms detected by the western blot assays might be the short cytoplasmic R monomeric soluble isoform, and the membranous one might be the longer N-extended AChE (R or S variants) and/or the AChE-S variant. To test this hypothesis, Reverse Transcription (RT) real-time PCR was performed. As seen in Figure 5, mRNA expression levels corresponding to the AChE-S isoform were low and did not change significantly after 15-, 30- or 60- min incubation with 3.5 mg/ml of glucose. However, mRNA expression transcripts tested for the AChE-R or the N-extended isoforms, were significantly (p < 0.05) up-regulated by 1.5 ± 0.15 and by 1.4 ± 0.14-fold respectively, compared to control Y79 cells.

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