Sialic acids linked to glycoconjugates of Fas regulate the caspase-9-dependent and mitochondria-mediated pathway of Fas-induced apoptosis in Jurkat T cell lymphoma.
To clarify the functions of sialic acids linked to glycoconjugates of Fas in Fas-induced apoptosis, Jurkat T cells, untreated and treated with neuraminidase, were incubated with anti-Fas monoclonal antibody, CH11. Apoptosis of Jurkat T cells induced by incubation with CH11 was enhanced by the pre-treatment with neuraminidase. By flow cytometry sialylated glycoconjugates were detected on the cell surface of Jurkat T cells using LFA lectin, which specifically reacts with sialic acid, and pre-treatment with Vibrio Cholerae neuraminidase resulted in desialylation of Jurkat cell surface glycoconjugates. The enhancement of Fas-induced apoptosis by pre-treatment with neuraminidase was inhibited by z-VAD-fmk, a broad caspase inhibitor, and Ac-LEHD-CHO, an inhibitor of caspase-9, but not by Ac-IETD-CHO an inhibitor of caspase-8 or 6, imipramine, an inhibitor of acidic sphingomyelinase, glutathione, an inhibitor of neutral sphingomyelinase and Fumonisin B1, an inhibitor of ceramide synthase. Mitochondrial membrane potentials (Deltapsim) measured with a Mitocapture assay kit demonstrated that the loss of Deltapsim involved in Fas-induced apoptosis was enhanced by pre-treatment with neuraminidase. Furthermore, Western blot analysis using polyclonal antibody (C-20) against Fas detected Fas at about 45 kDa, and pre-treatment with neuraminidase resulted in a reduction of the molecular weight of Fas of about 8 kDa. These data suggest that the enhancement of Fas-induced apoptosis by pre-treatment with neuraminidase was mediated by a caspase-9 dependent pathway closely associated with the loss of Deltapsim, not by activation of caspase-8, -6 or acidic and neutral sphingomyelinases, and that sialic acid linked to glycoconjugates of Fas may regulate Fas-induced apoptosis in human T cell lymphoma.
Suzuki
Osamu
O
School of Medicine, Department of Pathology, Fukushima Medical University, 1 Hikari-gaoka, Fukushima 960-1295, Japan. osuzuki@cc.fmu.ac.jp
Nozawa
Yoshihiro
Y
Abe
Masafumi
M
D017209:Apoptosis; D015153:Blotting, Western; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000096:biosynthesis* / Q000378:metabolism; D002470:Cell Survival; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D019169:Jurkat Cells; D037102:Lectins; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D009439:Neuraminidase / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012794:Sialic Acids / Q000378:metabolism / Q000502:physiology*; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism*
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D037102:Lectins; D012794:Sialic Acids; D019014:fas Receptor; D013108:Sphingomyelin Phosphodiesterase; D009439:Neuraminidase; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases