Cidlowski J A, King K L, Evans-Storms R B, Montague J W, Bortner C D, Hughes F M
Molecular Endocrinology Group, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Recent Prog Horm Res. 1996;51:457-90; discussion 490-1.
Apoptosis is a form of programmed cell death that occurs under numerous developmental and physiological conditions that require the selective elimination of cells from tissues and organs without the production of an inflammatory response. The initiation of apoptosis is controlled by a regulation of the balance between death and life signals perceived by the cell. A typical response of cells to an apoptotic stimulus includes a reduction in cell volume, compaction of intracellular organelles, chromatin condensation, and the generation of apoptotic bodies which contain degraded cellular components. Apoptotic bodies are often engulfed by neighboring cells or macrophages, preventing the occurrence of an inflammatory response in the region of the dying cells. Although the molecular basis for this cellular suicide is poorly understood, evidence indicates that apoptosis is an active process, requiring energy for its effective completion. We have sought to define the catabolic "effector" molecules that carry out the apoptotic process using glucocorticoid-induced apoptosis in rodent and human lymphocytes as model systems. These cells respond to dexamethasone with an arrest of cell growth, chromatin condensation, cell shrinkage, and the selective degradation of DNA, RNA, and protein. These effects are dependent on the presence of functional glucocorticoid receptors and require gene expression. The fragmentation of DNA and its associated cell shrinkage has been a focus of our efforts, because these effects reflect an irreversible commitment to death. Accordingly, we have developed assays to study apoptosis at the single cell level and to identify, purify, and clone the nuclease(s) that cause DNA damage in apoptotic cells. Using these approaches, we have identified and characterized a novel low molecular weight nuclease (NUC18) whose activity correlates with the DNA degradation occurring during apoptosis. NUC18 requires calcium for optimal activity in vitro and is inhibited by zinc and aurintricarboxylic acid, two known inhibitors of apoptosis. The amino acid sequence of pure NUC18 reveals a surprising homology to the cyclophilin family of proteins. Furthermore, recombinant cyclophilins have biochemical and pharmacological properties identical to those of NUC18. We have also studied the molecular basis for the catabolism of RNA and proteins that occurs during lymphocyte apoptosis. Recent experiments have identified selective cleavage of 28S ribosomal RNA and a novel nonlysosomal protease, both of which contribute to the demise of the cell. In summary, we present an evolving model that unifies the activation of apoptosis in lymphocytes by glucocorticoids with the counter-balancing effect of inhibitors such as Bcl-2.
细胞凋亡是一种程序性细胞死亡形式，发生在众多发育和生理条件下，这些条件需要从组织和器官中选择性清除细胞，且不产生炎症反应。细胞凋亡的启动由细胞所感知的死亡与生存信号之间的平衡调节所控制。细胞对凋亡刺激的典型反应包括细胞体积减小、细胞内细胞器致密化、染色质凝聚以及产生包含降解细胞成分的凋亡小体。凋亡小体常被邻近细胞或巨噬细胞吞噬，从而防止在垂死细胞区域发生炎症反应。尽管这种细胞自杀的分子基础仍知之甚少，但有证据表明细胞凋亡是一个活跃过程，有效完成该过程需要能量。我们试图利用啮齿动物和人类淋巴细胞中糖皮质激素诱导的细胞凋亡作为模型系统，来确定执行凋亡过程的分解代谢“效应器”分子。这些细胞对地塞米松的反应是细胞生长停滞、染色质凝聚、细胞收缩以及DNA、RNA和蛋白质的选择性降解。这些效应依赖于功能性糖皮质激素受体的存在且需要基因表达。DNA片段化及其相关的细胞收缩一直是我们研究的重点，因为这些效应反映了对死亡的不可逆承诺。因此，我们开发了在单细胞水平研究细胞凋亡的检测方法，并鉴定、纯化和克隆导致凋亡细胞中DNA损伤的核酸酶。利用这些方法，我们鉴定并表征了一种新型低分子量核酸酶（NUC18），其活性与凋亡过程中发生的DNA降解相关。NUC18在体外需要钙来实现最佳活性，并受到锌和金精三羧酸（两种已知的凋亡抑制剂）的抑制。纯NUC18的氨基酸序列显示出与亲环素蛋白家族惊人的同源性。此外，重组亲环素具有与NUC18相同的生化和药理特性。我们还研究了淋巴细胞凋亡过程中发生的RNA和蛋白质分解代谢的分子基础。最近的实验确定了28S核糖体RNA的选择性切割和一种新型非溶酶体蛋白酶，二者都促成了细胞的死亡。总之，我们提出了一个不断发展的模型，该模型将糖皮质激素对淋巴细胞凋亡的激活与诸如Bcl - 2等抑制剂的平衡作用统一起来。
