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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

Inhibiting AChE protein expression by different sh-RNA sequences reduces apoptosis in Y79 cells treated with glucose. (A) Y79 sh-RNA lenti-infected and selected cells (as described under Materials and Methods), were pre-treated in starvation medium containing 1% FBS and 1 mg/ml of glucose for 16–24 h and then treated with 3.5 mg/ml glucose for 1 h except for sh-control in lane 1. Cytoplasmic extracts were prepared and run for western blot analysis to detect AChE expression or anti α-Tubulin (for loading control) as described under Materials and Methods. The sample in lane 1 and those in 2–7 were run in separate lanes on the same gels. The western blot shown is one of two independent experiments. (B) Y79 sh-RNA lenti-infected and selected cells (as described under Materials and Methods), were pre-treated in starvation medium as described in A and then assayed by TUNEL (green) to detect apoptosis after 24 h in 3.5 mg/ml glucose. Nuclei were stained with DAPI (blue). Cells were photographed under an upright fluorescence microscope (Zeiss). Scale bar is 100 μm. (C) TUNEL positive Y79 sh-RNA lenti-infected and selected cells were counted and calculated as fold of control cells incubated under starvation medium, and plotted in histograms. Values are means ± SEM, (N = 3). *p < 0.05.
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Figure 6: Inhibiting AChE protein expression by different sh-RNA sequences reduces apoptosis in Y79 cells treated with glucose. (A) Y79 sh-RNA lenti-infected and selected cells (as described under Materials and Methods), were pre-treated in starvation medium containing 1% FBS and 1 mg/ml of glucose for 16–24 h and then treated with 3.5 mg/ml glucose for 1 h except for sh-control in lane 1. Cytoplasmic extracts were prepared and run for western blot analysis to detect AChE expression or anti α-Tubulin (for loading control) as described under Materials and Methods. The sample in lane 1 and those in 2–7 were run in separate lanes on the same gels. The western blot shown is one of two independent experiments. (B) Y79 sh-RNA lenti-infected and selected cells (as described under Materials and Methods), were pre-treated in starvation medium as described in A and then assayed by TUNEL (green) to detect apoptosis after 24 h in 3.5 mg/ml glucose. Nuclei were stained with DAPI (blue). Cells were photographed under an upright fluorescence microscope (Zeiss). Scale bar is 100 μm. (C) TUNEL positive Y79 sh-RNA lenti-infected and selected cells were counted and calculated as fold of control cells incubated under starvation medium, and plotted in histograms. Values are means ± SEM, (N = 3). *p < 0.05.

Mentions: In order to examine whether AChE expression plays a role in the hyperglycemia-induced apoptosis of Y79 cells, we tested five AChE shRNA sequences each cloned in pLKO.1- Lenti compatible vector (Sigma-Aldrich, St. Louis, USA). An empty pLKO.1 vector (Sigma-Aldrich, St. Louis, USA), devoid of a relevant shRNA sequence was used as control. As shown in Figure 6A, the expression of AChE tested under glucose exposure in the presence of the first sequences sh-1 (lane 3), was high and similar to that expressed in the presence of the sh-control-sequence (lane 2). The expression of AChE in these samples was considerably higher compared to sh-control Lenti-infected Y79 cells, which remained under 1 mg/ml glucose (lane1). These data indicate that sh-1 was not an efficient sequence for blocking AChE translation.


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)

Inhibiting AChE protein expression by different sh-RNA sequences reduces apoptosis in Y79 cells treated with glucose. (A) Y79 sh-RNA lenti-infected and selected cells (as described under Materials and Methods), were pre-treated in starvation medium containing 1% FBS and 1 mg/ml of glucose for 16–24 h and then treated with 3.5 mg/ml glucose for 1 h except for sh-control in lane 1. Cytoplasmic extracts were prepared and run for western blot analysis to detect AChE expression or anti α-Tubulin (for loading control) as described under Materials and Methods. The sample in lane 1 and those in 2–7 were run in separate lanes on the same gels. The western blot shown is one of two independent experiments. (B) Y79 sh-RNA lenti-infected and selected cells (as described under Materials and Methods), were pre-treated in starvation medium as described in A and then assayed by TUNEL (green) to detect apoptosis after 24 h in 3.5 mg/ml glucose. Nuclei were stained with DAPI (blue). Cells were photographed under an upright fluorescence microscope (Zeiss). Scale bar is 100 μm. (C) TUNEL positive Y79 sh-RNA lenti-infected and selected cells were counted and calculated as fold of control cells incubated under starvation medium, and plotted in histograms. Values are means ± SEM, (N = 3). *p < 0.05.
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Figure 6: Inhibiting AChE protein expression by different sh-RNA sequences reduces apoptosis in Y79 cells treated with glucose. (A) Y79 sh-RNA lenti-infected and selected cells (as described under Materials and Methods), were pre-treated in starvation medium containing 1% FBS and 1 mg/ml of glucose for 16–24 h and then treated with 3.5 mg/ml glucose for 1 h except for sh-control in lane 1. Cytoplasmic extracts were prepared and run for western blot analysis to detect AChE expression or anti α-Tubulin (for loading control) as described under Materials and Methods. The sample in lane 1 and those in 2–7 were run in separate lanes on the same gels. The western blot shown is one of two independent experiments. (B) Y79 sh-RNA lenti-infected and selected cells (as described under Materials and Methods), were pre-treated in starvation medium as described in A and then assayed by TUNEL (green) to detect apoptosis after 24 h in 3.5 mg/ml glucose. Nuclei were stained with DAPI (blue). Cells were photographed under an upright fluorescence microscope (Zeiss). Scale bar is 100 μm. (C) TUNEL positive Y79 sh-RNA lenti-infected and selected cells were counted and calculated as fold of control cells incubated under starvation medium, and plotted in histograms. Values are means ± SEM, (N = 3). *p < 0.05.
Mentions: In order to examine whether AChE expression plays a role in the hyperglycemia-induced apoptosis of Y79 cells, we tested five AChE shRNA sequences each cloned in pLKO.1- Lenti compatible vector (Sigma-Aldrich, St. Louis, USA). An empty pLKO.1 vector (Sigma-Aldrich, St. Louis, USA), devoid of a relevant shRNA sequence was used as control. As shown in Figure 6A, the expression of AChE tested under glucose exposure in the presence of the first sequences sh-1 (lane 3), was high and similar to that expressed in the presence of the sh-control-sequence (lane 2). The expression of AChE in these samples was considerably higher compared to sh-control Lenti-infected Y79 cells, which remained under 1 mg/ml glucose (lane1). These data indicate that sh-1 was not an efficient sequence for blocking AChE translation.

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