Rusnak J M, Calmels T P, Hoyt D G, Kondo Y, Yalowich J C, Lazo J S
Department of Pharmacology, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
Mol Pharmacol. 1996 Feb;49(2):244-52.
Higher order DNA fragmentation may be an essential signal in apoptosis. We found that etoposide (VP-16) induced apoptosis in human DU-145 prostatic carcinoma cells in a time- and concentration-dependent manner. Chromatin condensation was morphologically evident only when cells detached from the monolayer; untreated or VP-16-treated attached cells retained a normal morphology. We describe a radiolabeled alu-I sequence-based quantitative field inversion gel electrophoresis (QFIGE) method that permitted observation and quantification of discrete high molecular weight DNA fragments in detached (apoptotic) and attached (preapoptotic) DU-145 cells. The DNA fragments generated during the apoptotic death of these cells were > or = 1 (mega-base pairs) mbp, 450-600 (kilo-base pairs) kbp, and 30-50 kbp; we observed that these DNA fragments increased 9 +/- 2-, 8 +/- 2-, and 25 +/- 11-fold versus control, respectively, with a 24-hr exposure to 30 microM VP-16 in attached cell populations. In detached VP-16-treated cells, there was accrual of 30-50-kbp DNA fragments with a concomitant loss of the > or = 1-mbp and 450-600-kbp fragments; internucleosomal DNA cleavage was never observed. This pattern of high molecular weight DNA fragmentation was inhibited by cycloheximide treatment and was common to other apoptotic agents, including melphalan and bleomycin. These findings suggest that the > or = 1-mbp and 450-600-kbp DNA fragments are products of endonuclease activation and are not topoisomerase II/DNA interactions. Finally, the generation of the 30-50-kbp DNA fragments may mediate chromatin condensation, which characterizes apoptosis.
高阶DNA片段化可能是细胞凋亡中的一个重要信号。我们发现依托泊苷（VP - 16）能以时间和浓度依赖的方式诱导人DU - 145前列腺癌细胞凋亡。只有当细胞从单层脱离时，染色质凝聚在形态上才明显可见；未处理或经VP - 16处理的贴壁细胞保持正常形态。我们描述了一种基于放射性标记的alu - I序列的定量场反转凝胶电泳（QFIGE）方法，该方法能够观察和定量分离的（凋亡的）和贴壁的（凋亡前期的）DU - 145细胞中离散的高分子量DNA片段。这些细胞凋亡死亡过程中产生的DNA片段大小分别为≥1（兆碱基对）mbp、450 - 600（千碱基对）kbp和30 - 50 kbp；我们观察到，在贴壁细胞群体中，经24小时暴露于30μM VP - 16后，与对照组相比，这些DNA片段分别增加了9±2倍、8±2倍和25±11倍。在经VP - 16处理的脱离细胞中，30 - 50 kbp的DNA片段增加，同时≥1 - mbp和450 - 600 - kbp的片段减少；从未观察到核小体间DNA切割。这种高分子量DNA片段化模式受到环己酰亚胺处理的抑制，并且是包括美法仑和博来霉素在内的其他凋亡诱导剂所共有的。这些发现表明，≥1 - mbp和450 - 600 - kbp的DNA片段是内切核酸酶激活的产物，而非拓扑异构酶II/DNA相互作用的结果。最后，30 - 50 kbp DNA片段的产生可能介导了作为细胞凋亡特征的染色质凝聚。
