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Acetylation suppresses the proapoptotic activity of GD3 ganglioside.

Malisan F, Franchi L, Tomassini B, Ventura N, Condò I, Rippo MR, Rufini A, Liberati L, Nachtigall C, Kniep B, Testi R - J. Exp. Med. (2002)

Bottom Line: Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3.The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis.Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology and Signal Transduction, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, 00133 Rome, Italy.

ABSTRACT
GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3.

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Deacetylation of endogenous 9-O-acetyl GD3 induces apoptosis of U118 glioblastoma cells. (A) The expression of GD3 and 9-O-acetyl GD3 from U118 and U87 glioblastoma cells was analyzed by TLC-immunostaining. The percentage of apoptotic U118 (B) and U87 (C) cells was analyzed 24 h after transfection with empty vector (vector), dead mutant 9-O-acetylesterase (mEst), wild-type 9-O-acetylesterase (Est), and caspase-8 as a positive control. The data represent the mean ± 1 SD from five independent experiments. (D) 24 h after cotransfection with GFP-spectrin and empty vector (vector), or 9-O-acetylesterase (Est), U118 glioma cells were stained with anti-9-O acetyl GD3 mAb (open areas) or control antibody (shaded areas), and secondary RPE-conjugated antibody. The expression of 9-O-acetyl GD3 within electronically gated transfected cells was analyzed by flow cytometry. Data are representative of three independent experiments.
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fig5: Deacetylation of endogenous 9-O-acetyl GD3 induces apoptosis of U118 glioblastoma cells. (A) The expression of GD3 and 9-O-acetyl GD3 from U118 and U87 glioblastoma cells was analyzed by TLC-immunostaining. The percentage of apoptotic U118 (B) and U87 (C) cells was analyzed 24 h after transfection with empty vector (vector), dead mutant 9-O-acetylesterase (mEst), wild-type 9-O-acetylesterase (Est), and caspase-8 as a positive control. The data represent the mean ± 1 SD from five independent experiments. (D) 24 h after cotransfection with GFP-spectrin and empty vector (vector), or 9-O-acetylesterase (Est), U118 glioma cells were stained with anti-9-O acetyl GD3 mAb (open areas) or control antibody (shaded areas), and secondary RPE-conjugated antibody. The expression of 9-O-acetyl GD3 within electronically gated transfected cells was analyzed by flow cytometry. Data are representative of three independent experiments.

Mentions: To further support the notion that the ratio between GD3 and 9-O-acetyl GD3 might critically affect survival of tumor cells, we investigated tumor cell lines which constitutively synthesize and express both GD3 and 9-O-acetyl GD3. We tested the hypothesis that, by selectively breaking the endogenous GD3/9-O-acetyl GD3 balance in favor of GD3, we would induce apoptosis. The viral 9-O-acetylesterase is known to deacetylate 9-O-acetyl GD3 in neural tumor cells (18). Therefore, the viral 9-O-acetylesterase, and its catalytically dead mutant, were expressed in the human glioblastoma cells U118, which constitutively express both GD3 and 9-O-acetyl GD3, as assessed by TLC and immunostaining (Fig. 5 A). The 9-O-acetylesterase was also expressed in the human glioblastoma U87, which expresses GD3 but lacks detectable 9-O-acetyl GD3 (Fig. 5 A). As shown in Fig. 5 B, the 9-O-acetylesterase, but not its catalytically dead mutant, directly triggered apoptosis of U118 cells expressing 9-O-acetyl GD3. By contrast, the 9-O-acetylesterase, as well as its catalytically dead mutant, did not induce apoptosis in U87 cells lacking 9-O-acetyl GD3 (Fig. 5 C). To verify that the viral 9-O-acetylesterase was in fact active in glioblastoma cells, U118 cells were cotransfected with 9-O-acetylesterase and GFP-spectrin to allow the detection and electronic gating of transfected cells by FACS® analysis (24). Fig. 5 D shows that the levels of cellular 9-O-acetyl GD3 were significantly decreased in U118 cells transfected with the 9-O-acetylesterase, compared with vector-transfected cells. Together these results indicated that the 9-O-acetylesterase was specifically acting upon endogenous 9-O-acetyl GD3 and that the GD3/9-O-acetyl GD3 ratio may significantly control survival of tumor cells.


Acetylation suppresses the proapoptotic activity of GD3 ganglioside.

Malisan F, Franchi L, Tomassini B, Ventura N, Condò I, Rippo MR, Rufini A, Liberati L, Nachtigall C, Kniep B, Testi R - J. Exp. Med. (2002)

Deacetylation of endogenous 9-O-acetyl GD3 induces apoptosis of U118 glioblastoma cells. (A) The expression of GD3 and 9-O-acetyl GD3 from U118 and U87 glioblastoma cells was analyzed by TLC-immunostaining. The percentage of apoptotic U118 (B) and U87 (C) cells was analyzed 24 h after transfection with empty vector (vector), dead mutant 9-O-acetylesterase (mEst), wild-type 9-O-acetylesterase (Est), and caspase-8 as a positive control. The data represent the mean ± 1 SD from five independent experiments. (D) 24 h after cotransfection with GFP-spectrin and empty vector (vector), or 9-O-acetylesterase (Est), U118 glioma cells were stained with anti-9-O acetyl GD3 mAb (open areas) or control antibody (shaded areas), and secondary RPE-conjugated antibody. The expression of 9-O-acetyl GD3 within electronically gated transfected cells was analyzed by flow cytometry. Data are representative of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196066&req=5

fig5: Deacetylation of endogenous 9-O-acetyl GD3 induces apoptosis of U118 glioblastoma cells. (A) The expression of GD3 and 9-O-acetyl GD3 from U118 and U87 glioblastoma cells was analyzed by TLC-immunostaining. The percentage of apoptotic U118 (B) and U87 (C) cells was analyzed 24 h after transfection with empty vector (vector), dead mutant 9-O-acetylesterase (mEst), wild-type 9-O-acetylesterase (Est), and caspase-8 as a positive control. The data represent the mean ± 1 SD from five independent experiments. (D) 24 h after cotransfection with GFP-spectrin and empty vector (vector), or 9-O-acetylesterase (Est), U118 glioma cells were stained with anti-9-O acetyl GD3 mAb (open areas) or control antibody (shaded areas), and secondary RPE-conjugated antibody. The expression of 9-O-acetyl GD3 within electronically gated transfected cells was analyzed by flow cytometry. Data are representative of three independent experiments.
Mentions: To further support the notion that the ratio between GD3 and 9-O-acetyl GD3 might critically affect survival of tumor cells, we investigated tumor cell lines which constitutively synthesize and express both GD3 and 9-O-acetyl GD3. We tested the hypothesis that, by selectively breaking the endogenous GD3/9-O-acetyl GD3 balance in favor of GD3, we would induce apoptosis. The viral 9-O-acetylesterase is known to deacetylate 9-O-acetyl GD3 in neural tumor cells (18). Therefore, the viral 9-O-acetylesterase, and its catalytically dead mutant, were expressed in the human glioblastoma cells U118, which constitutively express both GD3 and 9-O-acetyl GD3, as assessed by TLC and immunostaining (Fig. 5 A). The 9-O-acetylesterase was also expressed in the human glioblastoma U87, which expresses GD3 but lacks detectable 9-O-acetyl GD3 (Fig. 5 A). As shown in Fig. 5 B, the 9-O-acetylesterase, but not its catalytically dead mutant, directly triggered apoptosis of U118 cells expressing 9-O-acetyl GD3. By contrast, the 9-O-acetylesterase, as well as its catalytically dead mutant, did not induce apoptosis in U87 cells lacking 9-O-acetyl GD3 (Fig. 5 C). To verify that the viral 9-O-acetylesterase was in fact active in glioblastoma cells, U118 cells were cotransfected with 9-O-acetylesterase and GFP-spectrin to allow the detection and electronic gating of transfected cells by FACS® analysis (24). Fig. 5 D shows that the levels of cellular 9-O-acetyl GD3 were significantly decreased in U118 cells transfected with the 9-O-acetylesterase, compared with vector-transfected cells. Together these results indicated that the 9-O-acetylesterase was specifically acting upon endogenous 9-O-acetyl GD3 and that the GD3/9-O-acetyl GD3 ratio may significantly control survival of tumor cells.

Bottom Line: Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3.The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis.Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology and Signal Transduction, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, 00133 Rome, Italy.

ABSTRACT
GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3.

Show MeSH
Related in: MedlinePlus