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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 Y79 cells treated by adding glucose or mannitol. Y79 cells were incubated for 16–24 h in starvation medium (1% FBS and 1 mg/ml glucose), and then glucose or mannitol was added (except in control) to reach a concentration of 3.5 mg/ml or 6 mg/ml and incubated for 1 or 2 h. (A) Nuclear extracts were prepared and run on SDS-PAGE, blotted, and probed with anti-PARP as described under Materials and Methods. (B) Three independent experiments were conducted and densitometry values were calculated for cleaved to non-cleaved ratios, and plotted in histograms as fold of control (cells cultured in starvation media containing 1% FBS and 1 mg/ml glucose). Values are means ± SEM, (N = 3). *p < 0.05.
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Figure 2: Cleavage of PARP in nuclear extracts of Y79 cells treated by adding glucose or mannitol. Y79 cells were incubated for 16–24 h in starvation medium (1% FBS and 1 mg/ml glucose), and then glucose or mannitol was added (except in control) to reach a concentration of 3.5 mg/ml or 6 mg/ml and incubated for 1 or 2 h. (A) Nuclear extracts were prepared and run on SDS-PAGE, blotted, and probed with anti-PARP as described under Materials and Methods. (B) Three independent experiments were conducted and densitometry values were calculated for cleaved to non-cleaved ratios, and plotted in histograms as fold of control (cells cultured in starvation media containing 1% FBS and 1 mg/ml glucose). Values are means ± SEM, (N = 3). *p < 0.05.

Mentions: Full-length PARP is an 116 kDa nuclear protein involved in the repair of DNA, in differentiation and in chromatin structure formation. During apoptosis this protein is cleaved by caspase-3, and possibly by other caspases, into an 89 kDa fragment (Lazebnik et al., 1994). Thus, demonstration of PARP cleavage is accepted as a molecular indicator for the induction of an apoptotic process. Since cleavage of PARP is an early stage (between 1 and 3 h) in apoptosis, we used western blot to measure its cleavage in nuclear extracts of cells prepared after 1 h incubation in different glycemic conditions as shown in Figure 2A. PARP cleavage was induced in cells exposed to 3.5 mg/ml or 6 mg/ml of glucose for 1 h, as compared to 1 mg/ml of glucose. To test a possible osmotic effect induced by hyperglycemia and leading to apoptosis, identical mannitol concentrations were added to starved-Y79 cells and PARP cleavage was examined after 1 h. We assessed quantitatively the magnitude of PARP cleavage and its significance from the cleaved/non-cleaved PARP ratio, as suggested by others (Biggs et al., 2001; Grader-Beck et al., 2007). We scanned the western blots and calculate the ratio of cleaved/non-cleaved PARP as shown in Figure 2B. PARP cleavage was similar in cells treated with 6 mg/ml of glucose or mannitol, but was more prominent, and statistically significant (p < 0.05), in cells treated with 3.5 mg/ml of glucose which showed 2.2 ± 0.16-fold of control, as compared to the same concentration of mannitol which showed 1.3 ± 0.22-fold of control. These results indicate that hyperglycemia caused by 6 mg/ml of glucose, induced apoptosis primarily as a result of hyperosmolarity, while at a lower concentration of 3.5 mg/ml of glucose, apoptosis was induced mainly as a result of hyperglycemia and to a lesser extent by hyperosmolarity.


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 Y79 cells treated by adding glucose or mannitol. Y79 cells were incubated for 16–24 h in starvation medium (1% FBS and 1 mg/ml glucose), and then glucose or mannitol was added (except in control) to reach a concentration of 3.5 mg/ml or 6 mg/ml and incubated for 1 or 2 h. (A) Nuclear extracts were prepared and run on SDS-PAGE, blotted, and probed with anti-PARP as described under Materials and Methods. (B) Three independent experiments were conducted and densitometry values were calculated for cleaved to non-cleaved ratios, and plotted in histograms as fold of control (cells cultured in starvation media containing 1% FBS and 1 mg/ml glucose). Values are means ± SEM, (N = 3). *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Cleavage of PARP in nuclear extracts of Y79 cells treated by adding glucose or mannitol. Y79 cells were incubated for 16–24 h in starvation medium (1% FBS and 1 mg/ml glucose), and then glucose or mannitol was added (except in control) to reach a concentration of 3.5 mg/ml or 6 mg/ml and incubated for 1 or 2 h. (A) Nuclear extracts were prepared and run on SDS-PAGE, blotted, and probed with anti-PARP as described under Materials and Methods. (B) Three independent experiments were conducted and densitometry values were calculated for cleaved to non-cleaved ratios, and plotted in histograms as fold of control (cells cultured in starvation media containing 1% FBS and 1 mg/ml glucose). Values are means ± SEM, (N = 3). *p < 0.05.
Mentions: Full-length PARP is an 116 kDa nuclear protein involved in the repair of DNA, in differentiation and in chromatin structure formation. During apoptosis this protein is cleaved by caspase-3, and possibly by other caspases, into an 89 kDa fragment (Lazebnik et al., 1994). Thus, demonstration of PARP cleavage is accepted as a molecular indicator for the induction of an apoptotic process. Since cleavage of PARP is an early stage (between 1 and 3 h) in apoptosis, we used western blot to measure its cleavage in nuclear extracts of cells prepared after 1 h incubation in different glycemic conditions as shown in Figure 2A. PARP cleavage was induced in cells exposed to 3.5 mg/ml or 6 mg/ml of glucose for 1 h, as compared to 1 mg/ml of glucose. To test a possible osmotic effect induced by hyperglycemia and leading to apoptosis, identical mannitol concentrations were added to starved-Y79 cells and PARP cleavage was examined after 1 h. We assessed quantitatively the magnitude of PARP cleavage and its significance from the cleaved/non-cleaved PARP ratio, as suggested by others (Biggs et al., 2001; Grader-Beck et al., 2007). We scanned the western blots and calculate the ratio of cleaved/non-cleaved PARP as shown in Figure 2B. PARP cleavage was similar in cells treated with 6 mg/ml of glucose or mannitol, but was more prominent, and statistically significant (p < 0.05), in cells treated with 3.5 mg/ml of glucose which showed 2.2 ± 0.16-fold of control, as compared to the same concentration of mannitol which showed 1.3 ± 0.22-fold of control. These results indicate that hyperglycemia caused by 6 mg/ml of glucose, induced apoptosis primarily as a result of hyperosmolarity, while at a lower concentration of 3.5 mg/ml of glucose, apoptosis was induced mainly as a result of hyperglycemia and to a lesser extent by hyperosmolarity.

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