Asher E, Payne C M, Bernstein C
Department of Pathology, University of Arizona, Tucson 85724, USA.
Leuk Lymphoma. 1995 Sep;19(1-2):107-19. doi: 10.3109/10428199509059664.
There is an extensive literature dating back to the late 1950's, on the damaging biological effects of radiolabeling DNA in vivo. Nonetheless, tritiated thymidine has often been used to label DNA in studies of programmed cell death (apoptosis). In the present study, we have investigated the effects of incorporation of tritiated thymidine into the DNA of an Epstein-Barr virus-transformed cell line (NC-37) in the absence of any other apoptosis-inducing agent. Cells were incubated in media containing 1-20 microCi/ml [methyl-3H]-thymidine ([3H]-TdR). At each concentration of tritiated thymidine used, cell proliferation ceased within 12 hours of incubation. The mode of cell death caused by tritiated thymidine incorporation was evaluated using DNA degradation patterns and cellular morphology. DNA degradation, in the absence of a "ladder" pattern, was shown by agarose gel electrophoresis. Electron microscopy was used as the "gold standard" to evaluate the specific morphologic type of cell death that accompanied the DNA degradation. Although some of the features of apoptosis were present, the cells lacked the early margination of the chromatin within an intact nucleus and surface blebbing leading to apoptotic body formation, two characteristic morphological features of apoptosis. We, therefore, coined the term "para-apoptosis" to be more precise about the morphologic type of cell death. The percent of para-apoptotic cells was quantitated by light microscopy using whole mount preparations (cytospins). The morphologic criteria of chromatin condensation, nuclear fragmentation, increase in cell density and cytoplasmic vacuolization were used for the evaluation of para-apoptosis by light microscopy of cytospin preparations. In the absence of tritiated thymidine, < 2% of the cells became apoptotic/para-apoptotic after 43 hours of incubation. However, at all concentrations of tritiated thymidine used in the incubation medium (1-20 microCi/ml), the number of para-apoptotic cells increased. In addition, we detected perturbations in the timing of the cell cycle of the surviving cells and an increase in the number of micronuclei after only one division cycle. The induction of para-apoptosis and micronuclei formation represent two distinct modes of cell death caused by tritiated thymidine incorporation. These studies emphasize the necessity for morphological examination in characterizing the induction of cell death in a new experimental system.
自20世纪50年代末以来，就有大量关于体内放射性标记DNA的有害生物学效应的文献。尽管如此，在程序性细胞死亡（凋亡）研究中，氚标记的胸腺嘧啶核苷常被用于标记DNA。在本研究中，我们研究了在没有任何其他凋亡诱导剂的情况下，将氚标记的胸腺嘧啶核苷掺入爱泼斯坦-巴尔病毒转化细胞系（NC-37）DNA中的效应。将细胞在含有1-20微居里/毫升[甲基-3H] -胸腺嘧啶核苷（[3H] -TdR）的培养基中孵育。在使用的每种氚标记胸腺嘧啶核苷浓度下，细胞增殖在孵育12小时内停止。使用DNA降解模式和细胞形态学评估由掺入氚标记胸腺嘧啶核苷引起的细胞死亡方式。琼脂糖凝胶电泳显示在没有“梯状”模式的情况下存在DNA降解。电子显微镜被用作“金标准”来评估伴随DNA降解的细胞死亡的特定形态学类型。尽管存在一些凋亡特征，但细胞缺乏完整细胞核内染色质的早期边缘化以及导致凋亡小体形成的表面起泡，这是凋亡的两个特征性形态学特征。因此，我们创造了“副凋亡”一词来更准确地描述细胞死亡的形态学类型。通过使用整装标本（细胞涂片）的光学显微镜对副凋亡细胞的百分比进行定量。染色质凝聚、核碎裂、细胞密度增加和细胞质空泡化的形态学标准用于通过细胞涂片制剂的光学显微镜评估副凋亡。在没有氚标记胸腺嘧啶核苷的情况下，孵育43小时后<2%的细胞发生凋亡/副凋亡。然而，在孵育培养基中使用的所有氚标记胸腺嘧啶核苷浓度（1-20微居里/毫升）下，副凋亡细胞的数量增加。此外，我们仅在一个分裂周期后就检测到存活细胞的细胞周期时间紊乱以及微核数量增加。副凋亡和微核形成的诱导代表了由掺入氚标记胸腺嘧啶核苷引起的两种不同的细胞死亡模式。这些研究强调了在新的实验系统中表征细胞死亡诱导时进行形态学检查的必要性。
