Indications for an active process underlying spontaneous and radiation-induced micronucleation in L929 cells

Purpose: To investigate the mechanism of micronucleus formation in irradiated L929 cells. Materials and methods: 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, Go6983) and after addition of N-acetylsphingosine (C-2-ceramide). PKC activity was determined by measuring the incorporation of [gamma-P-32]ATP into a suitable specific substrate. Proliferation was measured by detection of PCNA, RFP-A and BrdU (30-min pulse labelling) using both conventional immunoflourescence and laser scanning microscopy. Results: (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) MnT are characterized by a reduced transcription ability (PCNA, RFP-A). Conclusions: 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.