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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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Production of gangliosides in Niemann-Pick disease lymphoid cells upon treatment with anti-Fas or C6-ceramide.(a) Ganglioside production in Niemann-Pick lymphoblasts after treatment with anti-Fas antibody (200 ng/ml) for 15 min. Gangliosides were analyzed by HPTLC. The standard (Std) represents a mixture of bovine brain and melanoma gangliosides. For each cell line, the same number (50×106) of untreated and anti-Fas-treated cells was used. Lipids were extracted and loaded on the plate. (b) MS1418 cells (25×106) were incubated at 37°C for the indicated times with anti-Fas (500 ng/ml). Then, gangliosides were extracted and analyzed by HPTLC. Std represents standard gangliosides from melanoma (Mel.) or bovine brain. (c) Tre NPD lymphoblasts (25×106) cells were treated for 10 min at 37°C with 50 µM of C6-ceramide dissolved in ethanol. Then, gangliosides were purified and analyzed by HPTLC.
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pone-0019974-g001: Production of gangliosides in Niemann-Pick disease lymphoid cells upon treatment with anti-Fas or C6-ceramide.(a) Ganglioside production in Niemann-Pick lymphoblasts after treatment with anti-Fas antibody (200 ng/ml) for 15 min. Gangliosides were analyzed by HPTLC. The standard (Std) represents a mixture of bovine brain and melanoma gangliosides. For each cell line, the same number (50×106) of untreated and anti-Fas-treated cells was used. Lipids were extracted and loaded on the plate. (b) MS1418 cells (25×106) were incubated at 37°C for the indicated times with anti-Fas (500 ng/ml). Then, gangliosides were extracted and analyzed by HPTLC. Std represents standard gangliosides from melanoma (Mel.) or bovine brain. (c) Tre NPD lymphoblasts (25×106) cells were treated for 10 min at 37°C with 50 µM of C6-ceramide dissolved in ethanol. Then, gangliosides were purified and analyzed by HPTLC.

Mentions: The production of gangliosides was analyzed after Fas ligation by thin-layer chromatography. As illustrated in Fig. 1a, Niemann-Pick lymphoblasts (MS1418 and Tre cell lines) synthesized within 5 to 15 min several new species of gangliosides of the A-series (GM2, GM1, GD1a) with a whole ganglioside pattern reminiscent of that in brain [29]. Identification of gangliosides was carried out by immunostaining on thin-layer plates [26] after migration of the gangliosides, using a mixture of monoclonal antibodies known to react respectively with GM3, GM2 and GM1 as shown in Fig. 2. Identification of the other species, done by co-migration with authentic standards, indicated the presence of GD1a, GD1b, and GT1b, GD3 not being anymore the predominant ganglioside. An intriguing feature was the migration of these additional gangliosides as single bands (see also the lane of standards in Fig. 1a) instead of the doublets that can be seen with GM3 and GD3 as in untreated cells. Single bands of gangliosides from normal human tissues are usually seen only with brain and red cells. It should be noted that GM3 level was not affected by anti-Fas treatment, suggesting that the generation of complex gangliosides did not originate from the pre-existing GM3 pool (Fig. 1a).


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)

Production of gangliosides in Niemann-Pick disease lymphoid cells upon treatment with anti-Fas or C6-ceramide.(a) Ganglioside production in Niemann-Pick lymphoblasts after treatment with anti-Fas antibody (200 ng/ml) for 15 min. Gangliosides were analyzed by HPTLC. The standard (Std) represents a mixture of bovine brain and melanoma gangliosides. For each cell line, the same number (50×106) of untreated and anti-Fas-treated cells was used. Lipids were extracted and loaded on the plate. (b) MS1418 cells (25×106) were incubated at 37°C for the indicated times with anti-Fas (500 ng/ml). Then, gangliosides were extracted and analyzed by HPTLC. Std represents standard gangliosides from melanoma (Mel.) or bovine brain. (c) Tre NPD lymphoblasts (25×106) cells were treated for 10 min at 37°C with 50 µM of C6-ceramide dissolved in ethanol. Then, gangliosides were purified and analyzed by HPTLC.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101221&req=5

pone-0019974-g001: Production of gangliosides in Niemann-Pick disease lymphoid cells upon treatment with anti-Fas or C6-ceramide.(a) Ganglioside production in Niemann-Pick lymphoblasts after treatment with anti-Fas antibody (200 ng/ml) for 15 min. Gangliosides were analyzed by HPTLC. The standard (Std) represents a mixture of bovine brain and melanoma gangliosides. For each cell line, the same number (50×106) of untreated and anti-Fas-treated cells was used. Lipids were extracted and loaded on the plate. (b) MS1418 cells (25×106) were incubated at 37°C for the indicated times with anti-Fas (500 ng/ml). Then, gangliosides were extracted and analyzed by HPTLC. Std represents standard gangliosides from melanoma (Mel.) or bovine brain. (c) Tre NPD lymphoblasts (25×106) cells were treated for 10 min at 37°C with 50 µM of C6-ceramide dissolved in ethanol. Then, gangliosides were purified and analyzed by HPTLC.
Mentions: The production of gangliosides was analyzed after Fas ligation by thin-layer chromatography. As illustrated in Fig. 1a, Niemann-Pick lymphoblasts (MS1418 and Tre cell lines) synthesized within 5 to 15 min several new species of gangliosides of the A-series (GM2, GM1, GD1a) with a whole ganglioside pattern reminiscent of that in brain [29]. Identification of gangliosides was carried out by immunostaining on thin-layer plates [26] after migration of the gangliosides, using a mixture of monoclonal antibodies known to react respectively with GM3, GM2 and GM1 as shown in Fig. 2. Identification of the other species, done by co-migration with authentic standards, indicated the presence of GD1a, GD1b, and GT1b, GD3 not being anymore the predominant ganglioside. An intriguing feature was the migration of these additional gangliosides as single bands (see also the lane of standards in Fig. 1a) instead of the doublets that can be seen with GM3 and GD3 as in untreated cells. Single bands of gangliosides from normal human tissues are usually seen only with brain and red cells. It should be noted that GM3 level was not affected by anti-Fas treatment, suggesting that the generation of complex gangliosides did not originate from the pre-existing GM3 pool (Fig. 1a).

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