Azuma Yutaro, Sato Hirotaka, Higai Koji, Matsumoto Kojiro
Department of Clinical Chemistry, School of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan.
Biol Pharm Bull. 2007 Sep;30(9):1680-4. doi: 10.1248/bpb.30.1680.
We previously reported that, in Jurkat human T cells, the topoisomerase II inhibitor etoposide enhances sialidase activity and reduces cell surface sialic acid levels at an early stage of apoptosis and that the decreases in sialic acid are suppressed by the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid [Azuma Y., et al., Glycoconj. J., 17, 301-306 (2000)]. In the current studies, we treated Jurkat cells with etoposide and examined the changes in the cell surface levels of gangliosides GM1, GM2, GM3, GD1a, and GD3 at physiological pH using anti-ganglioside antibodies. We also examined the sialidase activity on the cell surface using 4-methylumbelliferyl N-acetylneuraminic acid and measured the mRNA expression of the plasma membrane-associated sialidase Neu3 and the lysozomal Neu1 using real-time PCR. We found an increase in GM3 and a decrease in GD3 during the early stage (4 h) of etoposide-induced apoptosis that preceded the increase in cell surface exposure of phosphatidylserine (4 to 6 h). The caspase 3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde significantly suppressed changes in GM3 and GD3 and blocked the enhanced cell surface sialidase activity. Furthermore, etoposide caused a gradual up-regulation of Neu3 mRNA expression but not Neu1 mRNA expression. Enhanced Neu3 mRNA expression was suppressed in the presence of caspase 3 inhibitor. These results indicate that Neu3 is up-regulated in Jurkat cells undergoing etoposide-induced apoptosis through intracellular signaling events downstream of caspase 3 activation and that enhanced Neu3 activity is closely related to the changes of cell surface ganglioside composition.
我们之前报道过，在人Jurkat T细胞中，拓扑异构酶II抑制剂依托泊苷在凋亡早期可增强唾液酸酶活性并降低细胞表面唾液酸水平，且唾液酸的减少可被唾液酸酶抑制剂2,3-脱氢-2-脱氧-N-乙酰神经氨酸所抑制[Azuma Y.等人，《糖缀合物杂志》，17, 301 - 306 (2000)]。在当前研究中，我们用依托泊苷处理Jurkat细胞，并使用抗神经节苷脂抗体在生理pH值下检测神经节苷脂GM1、GM2、GM3、GD1a和GD3的细胞表面水平变化。我们还使用4-甲基伞形酮基N-乙酰神经氨酸检测细胞表面的唾液酸酶活性，并通过实时PCR测量质膜相关唾液酸酶Neu3和溶酶体Neu1的mRNA表达。我们发现在依托泊苷诱导凋亡的早期阶段（4小时），GM3增加而GD3减少，这发生在磷脂酰丝氨酸细胞表面暴露增加（4至6小时）之前。半胱天冬酶3抑制剂乙酰-天冬氨酸-谷氨酸-缬氨酸-天冬氨酸醛可显著抑制GM3和GD3的变化，并阻断增强的细胞表面唾液酸酶活性。此外，依托泊苷导致Neu3 mRNA表达逐渐上调，但不影响Neu1 mRNA表达。在存在半胱天冬酶3抑制剂的情况下，增强的Neu3 mRNA表达受到抑制。这些结果表明，在经历依托泊苷诱导凋亡的Jurkat细胞中，Neu3通过半胱天冬酶3激活下游的细胞内信号事件而上调，且增强的Neu3活性与细胞表面神经节苷脂组成的变化密切相关。
