Uhlemann A C, Müller C, Madlung J, Gulbins E, Lang F
Dept. of Physiology I, University of Tübingen, Gmelinstrasse 5, 72076 Tübingen, Germany.
Cell Physiol Biochem. 2000;10(4):219-28. doi: 10.1159/000016353.
Apoptosis (programmed cell death) is an active physiological mechanism from which removal of abundant or potentially harmful cells follows. Apoptosis of lymphocytes is critical for the development of the immune system and during the immune response. As we have shown previously, moderate osmotic cell shrinkage interferes with CD95(Fas/Apo-1)-induced cell death. The present study has been performed to further elucidate the underlying mechanisms. To this end, apoptosis in Jurkat T-lymphocytes was elicited by triggering the CD95-receptor with monoclonal CD95/Fas-antibody. Osmotic cell shrinkage which was induced by the addition of 100 mM NaCl, did not significantly interfere with CD95-induced phosphatidylserine exposure nor the activation of caspase 3 activity as determined by PARP cleavage, DEVD-AMC consumption, or the activation of PAK2-kinase. However, osmotic cell shrinkage almost abolished CD95-induced DNA fragmentation (as revealed by propidium iodide staining) and the activation of a DNase as evidenced from SDS-PAGE gel assay. Western blot analysis showed CD95-induced tyrosine phosphorylation of a nuclear protein of ca. 20 kD which comigrated with nuclease activity. This tyrosine phosphorylation was almost completely abolished by the addition of 100 mM NaCl. Furthermore, osmotic cell shrinkage blunted the CD95-induced activation of the Src-like kinase p56lck. It is concluded that different signaling pathways mediate FITC-Annexin-V binding and DNase activation. Only the latter is sensitive to osmotic cell shrinkage.
凋亡（程序性细胞死亡）是一种主动的生理机制，通过该机制可清除多余的或潜在有害的细胞。淋巴细胞的凋亡对于免疫系统的发育以及免疫反应过程至关重要。正如我们之前所表明的，适度的渗透性细胞收缩会干扰CD95（Fas/Apo-1）诱导的细胞死亡。本研究旨在进一步阐明其潜在机制。为此，用单克隆CD95/Fas抗体触发CD95受体，从而诱导Jurkat T淋巴细胞发生凋亡。通过添加100 mM NaCl诱导的渗透性细胞收缩，并未显著干扰CD95诱导的磷脂酰丝氨酸暴露，也未干扰通过PARP裂解、DEVD-AMC消耗或PAK2激酶激活所测定的caspase 3活性。然而，渗透性细胞收缩几乎完全消除了CD95诱导的DNA片段化（通过碘化丙啶染色显示）以及SDS-PAGE凝胶分析所证实的DNase激活。蛋白质印迹分析显示，CD95诱导约20 kD的核蛋白发生酪氨酸磷酸化，该核蛋白与核酸酶活性共迁移。添加100 mM NaCl后，这种酪氨酸磷酸化几乎完全被消除。此外，渗透性细胞收缩减弱了CD95诱导的Src样激酶p56lck的激活。得出的结论是，不同的信号通路介导FITC-Annexin-V结合和DNase激活。只有后者对渗透性细胞收缩敏感。
