Weis M, Schlegel J, Kass G E, Holmström T H, Peters I, Eriksson J, Orrenius S, Chow S C
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
Exp Cell Res. 1995 Aug;219(2):699-708. doi: 10.1006/excr.1995.1281.
In the present study we investigated the Fas-mediated cellular events using the human leukemic T cell line, JURKAT. Ligation of the Fas receptor with a monoclonal antibody (IgM) resulted in the rapid (within 3 h) induction of apoptosis and was characterized by a sequence of distinct morphological and biochemical events. Thus, plasma membrane blebbing, condensation of the chromatin, and formation of high-molecular-weight (HMW) DNA fragments were the earliest events observed (by 45 min). They were followed by cleavage of DNA into oligonucleosomal-length fragments (laddering pattern) and the formation of apoptotic bodies, and finally, rounding of the apoptotic cells and complete cleavage of DNA into oligonucleosomal-length fragments occurred. The mitochondria remained structurally intact up to the stage of oligonucleosomal-length DNA cleavage, and the ability of the cells to exclude trypan blue was not compromised throughout the time course of the experiments. In contrast to many other model systems, apoptosis in JURKAT cells after anti-Fas treatment did not require the presence of extracellular Ca2+ or Mg2+ and was only partially inhibited by Zn2+. In addition, Fas-mediated apoptosis was unaffected by the presence of free radical scavengers or inhibitors of protein phosphatases, protein kinases, and nitric oxide synthesis. However, the serine protease inhibitors, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI) prevented anti-Fas-induced apoptosis in JURKAT cells. Low concentrations of these inhibitors blocked oligonucleosomal-length, but not HMW, DNA fragmentation. The latter required a higher concentration of TPCK or DCI to block. In addition, low concentrations of DCI also prevented Fas-mediated plasma membrane blebbing. In summary, our results suggest that proteolysis plays a central role in Fas-mediated apoptosis and that distinct proteolytic enzymes are involved in HMW DNA fragmentation, and oligonucleosomal-length DNA fragmentation, as well as in plasma membrane blebbing.
在本研究中，我们使用人白血病T细胞系JURKAT研究了Fas介导的细胞事件。用单克隆抗体（IgM）连接Fas受体导致快速（3小时内）诱导凋亡，并以一系列独特的形态学和生物化学事件为特征。因此，质膜起泡、染色质凝聚和高分子量（HMW）DNA片段的形成是最早观察到的事件（45分钟时）。随后DNA被切割成寡核小体长度的片段（梯状模式）并形成凋亡小体，最后，凋亡细胞变圆，DNA完全切割成寡核小体长度的片段。直到寡核小体长度的DNA切割阶段，线粒体在结构上仍保持完整，并且在整个实验过程中细胞排除台盼蓝的能力未受损害。与许多其他模型系统不同，抗Fas处理后JURKAT细胞中的凋亡不需要细胞外Ca2+或Mg2+的存在，并且仅被Zn2+部分抑制。此外，Fas介导的凋亡不受自由基清除剂或蛋白磷酸酶、蛋白激酶和一氧化氮合成抑制剂的存在的影响。然而，丝氨酸蛋白酶抑制剂N-对甲苯磺酰-L-苯丙氨酸氯甲基酮（TPCK）和3,4-二氯异香豆素（DCI）可阻止JURKAT细胞中抗Fas诱导的凋亡。低浓度的这些抑制剂可阻断寡核小体长度的DNA片段化，但不能阻断HMW DNA片段化。后者需要更高浓度的TPCK或DCI来阻断。此外，低浓度的DCI还可阻止Fas介导的质膜起泡。总之，我们的结果表明蛋白水解在Fas介导的凋亡中起核心作用，并且不同的蛋白水解酶参与HMW DNA片段化、寡核小体长度的DNA片段化以及质膜起泡。
