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Production of multiple brain-like ganglioside species is dispensable for fas-induced apoptosis of lymphoid cells.

Popa I, Therville N, Carpentier S, Levade T, Cuvillier O, Portoukalian J - PLoS ONE (2011)

Bottom Line: Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells.Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process.In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Recherche Dermatologique, EA4169 Université de Lyon-1, Hôpital Edouard Herriot, Lyon, France.

ABSTRACT
Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells. The present study was undertaken to clarify the role of this enzyme in the generation of gangliosides during apoptosis triggered by Fas ligation. The issue was addressed by using aSMase-deficient and aSMase-corrected cell lines derived from Niemann-Pick disease (NPD) patients. Fas cross-linking elicited a rapid production of large amounts of complex a- and b-series species of gangliosides with a pattern and a chromatographic behavior as single bands reminiscent of brain gangliosides. The gangliosides were synthesized within the first ten minutes and completely disappeared within thirty minutes after stimulation. Noteworthy is the observation that GD3 was not the only ganglioside produced. The production of gangliosides and the onset of apoptotic hallmarks occurred similarly in both aSMase-deficient and aSMase-corrected NPD lymphoid cells, indicating that aSMase activation is not accountable for ganglioside generation. Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process. In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.

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Pharmacological inhibition of ganglioside production does not prevent from anti-Fas-induced apoptosis.(a) Production of gangliosides in control lymphoblasts after treatment with 200 ng/ml anti-Fas antibody for 10 min with or without pre-treatment with 10 µM PDMP. Melanoma (Mel. Std) and bovine brain (Brain Std) ganglioside mixtures were used as standards. (b) Effect of PDMP on the production of gangliosides by NPD (MS1418) cells that were incubated as described in (a). (c) Effect of PDMP on the production of neutral glycosphingolipids by NPD (MS1418) cells that were incubated as described in (a). GlcCer, glucosylceramide; LacCer, lactosylceramide. (d) Control (Con) and NPD lymphoblasts were pre-incubated for 24 h with or without 10 µM PDMP or 400 µM DNJ, and treated without or with 100 ng/ml anti-Fas antibody for 5 h under serum-free conditions. Cell surface exposure of phosphatidylserine was determined by flow cytometry using annexin V-FITC/PI labelling (ns, not significant). (e) Control (Con), NPD (MS1418) and aSMase-transduced NPD (MS1418+) cells were incubated as indicated in (d) and cell extracts were subjected to 15% SDS-PAGE and immunoblotted with anti-caspase-3 antibody. Migrations indicated are: pro-caspase-3, full-length inactive caspase-3; p20 and p17, active subunits.
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pone-0019974-g003: Pharmacological inhibition of ganglioside production does not prevent from anti-Fas-induced apoptosis.(a) Production of gangliosides in control lymphoblasts after treatment with 200 ng/ml anti-Fas antibody for 10 min with or without pre-treatment with 10 µM PDMP. Melanoma (Mel. Std) and bovine brain (Brain Std) ganglioside mixtures were used as standards. (b) Effect of PDMP on the production of gangliosides by NPD (MS1418) cells that were incubated as described in (a). (c) Effect of PDMP on the production of neutral glycosphingolipids by NPD (MS1418) cells that were incubated as described in (a). GlcCer, glucosylceramide; LacCer, lactosylceramide. (d) Control (Con) and NPD lymphoblasts were pre-incubated for 24 h with or without 10 µM PDMP or 400 µM DNJ, and treated without or with 100 ng/ml anti-Fas antibody for 5 h under serum-free conditions. Cell surface exposure of phosphatidylserine was determined by flow cytometry using annexin V-FITC/PI labelling (ns, not significant). (e) Control (Con), NPD (MS1418) and aSMase-transduced NPD (MS1418+) cells were incubated as indicated in (d) and cell extracts were subjected to 15% SDS-PAGE and immunoblotted with anti-caspase-3 antibody. Migrations indicated are: pro-caspase-3, full-length inactive caspase-3; p20 and p17, active subunits.

Mentions: Interestingly, the aSMase-transduced NPD (line MS1418+) ganglioside pattern after anti-Fas treatment was comparable to that of the untransduced MS1418 NPD cell line (Fig. 1a). The ganglioside generation was quantitatively very important, up to 50-fold above the control, but it was transient as most of the neosynthesized gangliosides disappeared after 30 min and the ganglioside pattern was back to normal within 60 min beyond treatment (Fig. 1b). In addition, ganglioside production was also observed when cells were treated with exogenous C6-ceramide (Fig. 1c). An interesting observation is that, after anti-Fas or cell-permeable ceramide treatment, EBV-transformed lymphoblasts derived from control subjects showed a rapid synthesis of gangliosides, but the increase involved mostly the GM3 and GD3 ganglioside species already found in the control cells, in striking contrast with the ganglioside profile seen with NPD cells (see Fig. 3a). This different response in the ganglioside species neosynthesized between control and aSMase-deficient (but also aSMase-transduced) cell lines is not yet understood. It is noteworthy to point out that increased levels of the lactosylceramide precursor were seen concomitantly with the rapid increase in ganglioside biosynthesis (see Fig. 3c).


Production of multiple brain-like ganglioside species is dispensable for fas-induced apoptosis of lymphoid cells.

Popa I, Therville N, Carpentier S, Levade T, Cuvillier O, Portoukalian J - PLoS ONE (2011)

Pharmacological inhibition of ganglioside production does not prevent from anti-Fas-induced apoptosis.(a) Production of gangliosides in control lymphoblasts after treatment with 200 ng/ml anti-Fas antibody for 10 min with or without pre-treatment with 10 µM PDMP. Melanoma (Mel. Std) and bovine brain (Brain Std) ganglioside mixtures were used as standards. (b) Effect of PDMP on the production of gangliosides by NPD (MS1418) cells that were incubated as described in (a). (c) Effect of PDMP on the production of neutral glycosphingolipids by NPD (MS1418) cells that were incubated as described in (a). GlcCer, glucosylceramide; LacCer, lactosylceramide. (d) Control (Con) and NPD lymphoblasts were pre-incubated for 24 h with or without 10 µM PDMP or 400 µM DNJ, and treated without or with 100 ng/ml anti-Fas antibody for 5 h under serum-free conditions. Cell surface exposure of phosphatidylserine was determined by flow cytometry using annexin V-FITC/PI labelling (ns, not significant). (e) Control (Con), NPD (MS1418) and aSMase-transduced NPD (MS1418+) cells were incubated as indicated in (d) and cell extracts were subjected to 15% SDS-PAGE and immunoblotted with anti-caspase-3 antibody. Migrations indicated are: pro-caspase-3, full-length inactive caspase-3; p20 and p17, active subunits.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101221&req=5

pone-0019974-g003: Pharmacological inhibition of ganglioside production does not prevent from anti-Fas-induced apoptosis.(a) Production of gangliosides in control lymphoblasts after treatment with 200 ng/ml anti-Fas antibody for 10 min with or without pre-treatment with 10 µM PDMP. Melanoma (Mel. Std) and bovine brain (Brain Std) ganglioside mixtures were used as standards. (b) Effect of PDMP on the production of gangliosides by NPD (MS1418) cells that were incubated as described in (a). (c) Effect of PDMP on the production of neutral glycosphingolipids by NPD (MS1418) cells that were incubated as described in (a). GlcCer, glucosylceramide; LacCer, lactosylceramide. (d) Control (Con) and NPD lymphoblasts were pre-incubated for 24 h with or without 10 µM PDMP or 400 µM DNJ, and treated without or with 100 ng/ml anti-Fas antibody for 5 h under serum-free conditions. Cell surface exposure of phosphatidylserine was determined by flow cytometry using annexin V-FITC/PI labelling (ns, not significant). (e) Control (Con), NPD (MS1418) and aSMase-transduced NPD (MS1418+) cells were incubated as indicated in (d) and cell extracts were subjected to 15% SDS-PAGE and immunoblotted with anti-caspase-3 antibody. Migrations indicated are: pro-caspase-3, full-length inactive caspase-3; p20 and p17, active subunits.
Mentions: Interestingly, the aSMase-transduced NPD (line MS1418+) ganglioside pattern after anti-Fas treatment was comparable to that of the untransduced MS1418 NPD cell line (Fig. 1a). The ganglioside generation was quantitatively very important, up to 50-fold above the control, but it was transient as most of the neosynthesized gangliosides disappeared after 30 min and the ganglioside pattern was back to normal within 60 min beyond treatment (Fig. 1b). In addition, ganglioside production was also observed when cells were treated with exogenous C6-ceramide (Fig. 1c). An interesting observation is that, after anti-Fas or cell-permeable ceramide treatment, EBV-transformed lymphoblasts derived from control subjects showed a rapid synthesis of gangliosides, but the increase involved mostly the GM3 and GD3 ganglioside species already found in the control cells, in striking contrast with the ganglioside profile seen with NPD cells (see Fig. 3a). This different response in the ganglioside species neosynthesized between control and aSMase-deficient (but also aSMase-transduced) cell lines is not yet understood. It is noteworthy to point out that increased levels of the lactosylceramide precursor were seen concomitantly with the rapid increase in ganglioside biosynthesis (see Fig. 3c).

Bottom Line: Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells.Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process.In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Recherche Dermatologique, EA4169 Université de Lyon-1, Hôpital Edouard Herriot, Lyon, France.

ABSTRACT
Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells. The present study was undertaken to clarify the role of this enzyme in the generation of gangliosides during apoptosis triggered by Fas ligation. The issue was addressed by using aSMase-deficient and aSMase-corrected cell lines derived from Niemann-Pick disease (NPD) patients. Fas cross-linking elicited a rapid production of large amounts of complex a- and b-series species of gangliosides with a pattern and a chromatographic behavior as single bands reminiscent of brain gangliosides. The gangliosides were synthesized within the first ten minutes and completely disappeared within thirty minutes after stimulation. Noteworthy is the observation that GD3 was not the only ganglioside produced. The production of gangliosides and the onset of apoptotic hallmarks occurred similarly in both aSMase-deficient and aSMase-corrected NPD lymphoid cells, indicating that aSMase activation is not accountable for ganglioside generation. Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process. In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.

Show MeSH
Related in: MedlinePlus