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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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Control and Niemann-Pick disease lymphoblasts are equally sensitive to anti-Fas-induced apoptosis.(a) Control, NPD (MS1418 and Tre cell lines), and aSMase-transduced NPD (MS1418+ cell line) lymphoblasts were incubated in serum-free conditions with or without 100 ng/ml anti-Fas for 5 h. The percentage of apoptotic cells was evaluated by the DNA-specific fluorochrome Hoechst 33258. Nuclei were visualized by fluorescence microscopy, and a minimum of 300 cells were scored. Mean values and standard deviations for each sample from at least three different experiments are shown. (b) DEVDase activity was measured with the fluorogenic substrate Ac-DEVD-AMC in extracts from NPD (MS1418 and Tre cell lines), and the aSMase-transduced NPD (MS1418+ cell line) lymphoblasts treated for the indicated times without (open squares) or with 100 ng/ml anti-Fas antibody (filled squares). Results are means ± S.D. of at least three independent experiments. (c) Extracts from control, NPD (MS1418 and Tre cell lines), and aSMase-transduced NPD (MS1418+ cell line) lymphoblasts treated with anti-Fas antibody for 5 h 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-g004: Control and Niemann-Pick disease lymphoblasts are equally sensitive to anti-Fas-induced apoptosis.(a) Control, NPD (MS1418 and Tre cell lines), and aSMase-transduced NPD (MS1418+ cell line) lymphoblasts were incubated in serum-free conditions with or without 100 ng/ml anti-Fas for 5 h. The percentage of apoptotic cells was evaluated by the DNA-specific fluorochrome Hoechst 33258. Nuclei were visualized by fluorescence microscopy, and a minimum of 300 cells were scored. Mean values and standard deviations for each sample from at least three different experiments are shown. (b) DEVDase activity was measured with the fluorogenic substrate Ac-DEVD-AMC in extracts from NPD (MS1418 and Tre cell lines), and the aSMase-transduced NPD (MS1418+ cell line) lymphoblasts treated for the indicated times without (open squares) or with 100 ng/ml anti-Fas antibody (filled squares). Results are means ± S.D. of at least three independent experiments. (c) Extracts from control, NPD (MS1418 and Tre cell lines), and aSMase-transduced NPD (MS1418+ cell line) lymphoblasts treated with anti-Fas antibody for 5 h 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: We next compared the sensitivity to apoptosis of the NPD lymphoid cells. As we have previously reported [15], treatment with anti-Fas antibody induced apoptosis equally well in control, NPD (MS1418 and Tre cell lines), and corrected NPD MS1418+ cell line (Fig. 4a). We also examined whether aSMase influenced processing of executioner caspases (such as caspase-3 and -7), the proteases that drive the effector phase of apoptosis, and which are activated during Fas-induced apoptosis. To monitor executioner caspase activity, we utilized the fluorogenic substrate, Ac-DEVD-AMC, which corresponds to the cleavage site found in various caspase-3 and -7 targets. In control, NPD MS1418 and corrected NPD MS1418+ cell lines, we observed similar time-dependent increases in DEVDase activity (Fig. 4b) that correlated with the onset of apoptosis. Proteolytic cleavage of caspase-3 was also examined by Western blotting, and Fig. 4c illustrates the processing of caspase-3 into its respective active forms regardless of the aSMase status of the cell lines used. These findings clearly indicate that normal and NPD lymphoblast cells are equally sensitive to Fas-induced apoptosis. Similar results were found when cells were treated with exogenous C6-ceramide (data not shown).


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)

Control and Niemann-Pick disease lymphoblasts are equally sensitive to anti-Fas-induced apoptosis.(a) Control, NPD (MS1418 and Tre cell lines), and aSMase-transduced NPD (MS1418+ cell line) lymphoblasts were incubated in serum-free conditions with or without 100 ng/ml anti-Fas for 5 h. The percentage of apoptotic cells was evaluated by the DNA-specific fluorochrome Hoechst 33258. Nuclei were visualized by fluorescence microscopy, and a minimum of 300 cells were scored. Mean values and standard deviations for each sample from at least three different experiments are shown. (b) DEVDase activity was measured with the fluorogenic substrate Ac-DEVD-AMC in extracts from NPD (MS1418 and Tre cell lines), and the aSMase-transduced NPD (MS1418+ cell line) lymphoblasts treated for the indicated times without (open squares) or with 100 ng/ml anti-Fas antibody (filled squares). Results are means ± S.D. of at least three independent experiments. (c) Extracts from control, NPD (MS1418 and Tre cell lines), and aSMase-transduced NPD (MS1418+ cell line) lymphoblasts treated with anti-Fas antibody for 5 h 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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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101221&req=5

pone-0019974-g004: Control and Niemann-Pick disease lymphoblasts are equally sensitive to anti-Fas-induced apoptosis.(a) Control, NPD (MS1418 and Tre cell lines), and aSMase-transduced NPD (MS1418+ cell line) lymphoblasts were incubated in serum-free conditions with or without 100 ng/ml anti-Fas for 5 h. The percentage of apoptotic cells was evaluated by the DNA-specific fluorochrome Hoechst 33258. Nuclei were visualized by fluorescence microscopy, and a minimum of 300 cells were scored. Mean values and standard deviations for each sample from at least three different experiments are shown. (b) DEVDase activity was measured with the fluorogenic substrate Ac-DEVD-AMC in extracts from NPD (MS1418 and Tre cell lines), and the aSMase-transduced NPD (MS1418+ cell line) lymphoblasts treated for the indicated times without (open squares) or with 100 ng/ml anti-Fas antibody (filled squares). Results are means ± S.D. of at least three independent experiments. (c) Extracts from control, NPD (MS1418 and Tre cell lines), and aSMase-transduced NPD (MS1418+ cell line) lymphoblasts treated with anti-Fas antibody for 5 h 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: We next compared the sensitivity to apoptosis of the NPD lymphoid cells. As we have previously reported [15], treatment with anti-Fas antibody induced apoptosis equally well in control, NPD (MS1418 and Tre cell lines), and corrected NPD MS1418+ cell line (Fig. 4a). We also examined whether aSMase influenced processing of executioner caspases (such as caspase-3 and -7), the proteases that drive the effector phase of apoptosis, and which are activated during Fas-induced apoptosis. To monitor executioner caspase activity, we utilized the fluorogenic substrate, Ac-DEVD-AMC, which corresponds to the cleavage site found in various caspase-3 and -7 targets. In control, NPD MS1418 and corrected NPD MS1418+ cell lines, we observed similar time-dependent increases in DEVDase activity (Fig. 4b) that correlated with the onset of apoptosis. Proteolytic cleavage of caspase-3 was also examined by Western blotting, and Fig. 4c illustrates the processing of caspase-3 into its respective active forms regardless of the aSMase status of the cell lines used. These findings clearly indicate that normal and NPD lymphoblast cells are equally sensitive to Fas-induced apoptosis. Similar results were found when cells were treated with exogenous C6-ceramide (data not shown).

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