Abend M, Frombeck S, Van Beuningen D
Institute of Radiobiology, Federal Armed Forces Medical Academy, Munich, Germany.
Int J Radiat Biol. 1999 Dec;75(12):1567-78. doi: 10.1080/095530099139179.
To investigate the mechanism of micronucleus formation in irradiated L929 cells.
Radiation-induced micronuclei (MN) of L929 cells isolated at 48 and 72 h after irradiation were processed for detection of DNA-laddering and higher-order chromatin fragments using conventional gel electrophoresis and pulse-field gel electrophoresis. Quantification of double-strand breaks in micronuclei and nuclei was performed with the TdT assay and quantified using image analysis. The number of binucleated cells containing micronuclei (cytochalasin B method) was counted after application of three unspecific endonuclease inhibitors (aurin, ATA, spermine), a topoisomerase II inhibitor (VM-26), administration of two PKC inhibitors (H-7, Gö6983) and after addition of N-acetylsphingosine (C2-ceramide). PKC activity was determined by measuring the incorporation of [gamma-32P]ATP into a suitable specific substrate. Proliferation was measured by detection of PCNA, RFP-A and BrdU (30-min pulse labelling) using both conventional immunofluorescence and laser scanning microscopy.
(1) Higher chromatin fragments accumulated in MN with a size as they occur during early stages of apoptosis; (2) the frequency of MN was influenced by drugs known to play an important role in signalling and execution of apoptosis (endonucleases, topoisomerase II, protein kinases, ceramide); (3) MN are characterized by a reduced transcription ability (PCNA, RFP-A).
A proportion of L929 MN may be formed by an active process comparable with the early stages of apoptosis; it may play a role in the re-organization of the damaged genome.
研究受辐照L929细胞中微核形成的机制。
对辐照后48小时和72小时分离出的L929细胞的辐射诱导微核（MN）进行处理，使用常规凝胶电泳和脉冲场凝胶电泳检测DNA梯状条带和高阶染色质片段。采用TdT法对微核和细胞核中的双链断裂进行定量，并通过图像分析进行量化。在应用三种非特异性核酸内切酶抑制剂（金精三羧酸、ATA、精胺）、一种拓扑异构酶II抑制剂（VM - 26）、两种蛋白激酶C抑制剂（H - 7、Gö6983）后，以及添加N - 乙酰鞘氨醇（C2 - 神经酰胺）后，计数含有微核的双核细胞数量（细胞松弛素B法）。通过测量[γ - 32P]ATP掺入合适的特异性底物来测定蛋白激酶C活性。使用常规免疫荧光和激光扫描显微镜通过检测增殖细胞核抗原（PCNA）、红色荧光蛋白A（RFP - A）和溴脱氧尿苷（30分钟脉冲标记）来测量增殖情况。
（1）在微核中积累了更高的染色质片段，其大小与凋亡早期出现的片段相同；（2）微核频率受到已知在凋亡信号传导和执行中起重要作用的药物（核酸内切酶、拓扑异构酶II、蛋白激酶、神经酰胺）的影响；（3）微核的特征是转录能力降低（PCNA、RFP - A）。
一部分L929微核可能通过与凋亡早期相当的活跃过程形成；它可能在受损基因组的重组中起作用。
