Li X, Darzynkiewicz Z
Brander Cancer Research Institute, New York Medical College, Valhalla, New York 10595, USA.
Exp Cell Res. 2000 Feb 25;255(1):125-32. doi: 10.1006/excr.1999.4796.
Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a target of caspases during apoptosis: its cleavage onto 89- and 24-kDa fragments is considered to be a hallmark of the apoptotic mode of cell death. Another hallmark is the activation of endonuclease which targets internucleosomal DNA. The aim of the present study was to reveal cell cycle phase specificity as well as the temporal and sequence relationships of PARP cleavage vis-à-vis DNA fragmentation in two model systems of apoptosis, one induced by DNA damage via cell treatment with camptothecin (CPT) (mitochondria-induced pathway) and another by the cytotoxic ligand tumor necrosis factor alpha (TNF-alpha) (cell surface death receptor pathway). PARP cleavage was detected immunocytochemically using antibody which recognizes its 89-kDa fragment (PARP p89) while DNA fragmentation was assayed by in situ labeling of DNA strand breaks. The frequency and extent of PARP cleavage as well as DNA fragmentation were measured by mutiparameter flow and laser scanning cytometry. PARP cleavage, selective to S phase cells, was detected 90 min after administration of CPT. PARP cleavage in the cells treated with TNF-alpha was not selective to any cell cycle phase and was seen already after 30 min. DNA fragmentation trailed PARP cleavage by about 30 min and showed a similar pattern of cell cycle specificity. PARP p89 was present in nuclear chromatin but at least in the early phase of apoptosis it did not colocalize with DNA strand breaks. The rate of cleavage of PARP molecules in individual cells whether induced by CPT or TNF-alpha was rapid as reflected by the paucity of cells with a mixture of cleaved and noncleaved PARP molecules. In contrast, DNA fragmentation proceeded stepwise before reaching the maximal number of DNA strand breaks. Although time windows for PARP cleavage vs DNA fragmentation were different at early stages of apoptosis, a good overall correlation between the cytometric assays of apoptotic cells identification based on these events was observed in both CPT- and TNF-alpha-treated cultures.
聚（ADP - 核糖）聚合酶（PARP）是一种参与DNA修复的核酶，在细胞凋亡过程中是半胱天冬酶的作用靶点：其切割成89 kDa和24 kDa片段被认为是细胞凋亡死亡模式的一个标志。另一个标志是靶向核小体间DNA的核酸内切酶的激活。本研究的目的是揭示在两种细胞凋亡模型系统中PARP切割相对于DNA片段化的细胞周期阶段特异性以及时间和序列关系，一种是通过喜树碱（CPT）处理细胞诱导DNA损伤（线粒体诱导途径），另一种是通过细胞毒性配体肿瘤坏死因子α（TNF - α）（细胞表面死亡受体途径）。使用识别其89 kDa片段（PARP p89）的抗体通过免疫细胞化学检测PARP切割，而通过DNA链断裂的原位标记检测DNA片段化。通过多参数流式细胞术和激光扫描细胞术测量PARP切割的频率和程度以及DNA片段化。对S期细胞具有选择性的PARP切割在给予CPT后90分钟被检测到。用TNF - α处理的细胞中的PARP切割对任何细胞周期阶段都没有选择性，在30分钟后就可见。DNA片段化比PARP切割滞后约30分钟，并显示出类似的细胞周期特异性模式。PARP p89存在于核染色质中，但至少在细胞凋亡的早期阶段它与DNA链断裂不共定位。无论是由CPT还是TNF - α诱导，单个细胞中PARP分子的切割速率都很快，这反映在具有切割和未切割PARP分子混合物的细胞数量很少。相比之下，DNA片段化在达到最大数量的DNA链断裂之前是逐步进行的。虽然在细胞凋亡早期PARP切割与DNA片段化的时间窗口不同，但在CPT和TNF - α处理的培养物中，基于这些事件的凋亡细胞识别的细胞计数分析之间都观察到了良好的总体相关性。
