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Indications For An Active Process Underlying Spontaneous And Radiation-induced Micronucleation In L929 Cells.
M. Abend, S. Frombeck, D. van Beuningen
Published 1999 · Biology, Medicine
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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, 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. 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) MN 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.
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