Khodarev N N, Ashwell J D
Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1152, USA.
J Immunol. 1996 Feb 1;156(3):922-31.
Apoptotic cell death is typically accompanied by internucleosomal chromatin fragmentation. Although a number of candidate enzymes have been proposed, there is as yet no direct evidence for the involvement of any particular endonuclease in this process. Here we demonstrate the existence of an endonuclease(s) that is up-regulated during apoptotic T cell death. The endonuclease(s) is located in the nucleus, and its activity is increased up to eightfold by a variety of stimuli or conditions that induce apoptosis in T cell hybridomas and thymocytes. Treatments that prevent TCR-mediated apoptosis, such as cyclosporin A or concomitant administration of glucocorticoids, also prevent the induction of enzyme activity. The endonuclease activity is associated with three molecular forms, designated A, B, and C, with apparent M(r) of 49K, 47K, and 45K, respectively, and constitutes the major endonuclease activity in T hybridoma cells. From A exists in resting cells, and its activity is increased threefold after the induction of apoptosis. Forms B and C are absent in resting cells and are induced up to 20-fold after stimuli that lead to apoptosis. All three forms are Ca2+/Mg2+ dependent and are inhibited by Zn2+. This enzyme(s) introduces double strand breaks and single strand nicks into supercoiled plasmid DNA, demonstrating the mode of DNA fragmentation characteristic of products of apoptotic chromatin degradation. The enzyme(s) produces DNA fragments with 5'-P and 3'-OH terminals, also consistent with apoptotic chromatin degradation. Finally, enzyme solubilized from cells activated to die cleaves chromatin in nuclei isolated from unstimulated T hybridoma cells, yielding the classic DNA ladder. Because of its biologic properties, we named this enzyme(s) inducible lymphocyte Ca2+/Mg(2+)-dependent endonuclease, or ILCME. Because inducible lymphocyte Ca2+/Mg(2+)-dependent endonuclease possesses the key features predicted for an apoptosis-specific enzyme, it is a new candidate for an enzyme(s) that participates in DNA cleavage in apoptotic T cells.
凋亡性细胞死亡通常伴随着核小体间染色质片段化。尽管已经提出了许多候选酶，但尚无直接证据表明任何特定的核酸内切酶参与了这一过程。在此，我们证明了在凋亡的T细胞死亡过程中上调的一种核酸内切酶的存在。该核酸内切酶位于细胞核中，在多种诱导T细胞杂交瘤和胸腺细胞凋亡的刺激或条件下，其活性可增加至八倍。阻止TCR介导的凋亡的处理，如环孢菌素A或同时给予糖皮质激素，也可阻止酶活性的诱导。核酸内切酶活性与三种分子形式相关，分别命名为A、B和C，其表观分子量分别为49K、47K和45K，并且构成T杂交瘤细胞中的主要核酸内切酶活性。形式A存在于静息细胞中，凋亡诱导后其活性增加三倍。形式B和C在静息细胞中不存在，在导致凋亡的刺激后诱导增加至20倍。所有三种形式均依赖Ca2+/Mg2+，并被Zn2+抑制。这种酶将双链断裂和单链切口引入超螺旋质粒DNA，证明了凋亡染色质降解产物特有的DNA片段化模式。该酶产生具有5'-P和3'-OH末端的DNA片段，也与凋亡染色质降解一致。最后，从激活死亡的细胞中溶解的酶切割从未刺激的T杂交瘤细胞分离的细胞核中的染色质，产生经典的DNA梯带。由于其生物学特性，我们将这种酶命名为诱导性淋巴细胞Ca2+/Mg(2+)-依赖性核酸内切酶，或ILCME。由于诱导性淋巴细胞Ca2+/Mg(2+)-依赖性核酸内切酶具有凋亡特异性酶所预测的关键特征，它是参与凋亡T细胞中DNA切割的一种酶的新候选者。
