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

• Published in: International journal of radiation biology 1999, Vol.75 (12), p.1567-1578
• Main Author: Abend, S. Frombeck, D. Van Beuningen, M.
• Format: Article
• Language: English
• Subjects: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine - pharmacology; Animals; Aurintricarboxylic Acid - pharmacology; Benzhydryl Compounds - pharmacology; Biological and medical sciences; Bromodeoxyuridine - metabolism; Cell Division - physiology; Cell Division - radiation effects; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors; DNA, Neoplasm - metabolism; DNA, Neoplasm - radiation effects; Effects of various physical factors on living matter (vibrations, electric field, ultrasound, sound...); Electrophoresis, Gel, Pulsed-Field; Endonucleases - antagonists & inhibitors; Enzyme Inhibitors - pharmacology; Fibrosarcoma; Fundamental and applied biological sciences. Psychology; Mice; Micronuclei, Chromosome-Defective - radiation effects; Micronucleus Tests; Proliferating Cell Nuclear Antigen - biosynthesis; Proliferating Cell Nuclear Antigen - metabolism; Protein Kinase C - antagonists & inhibitors; Space life sciences; Spermine - pharmacology; Sphingosine - analogs & derivatives; Sphingosine - pharmacology; Teniposide - pharmacology; Tissues, organs and organisms biophysics; Topoisomerase II Inhibitors; Transcription, Genetic - radiation effects; Tumor Cells, Cultured - radiation effects
• ISSN: 0955-3002; 1362-3095
• DOI: 10.1080/095530099139179

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 72h 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 (C2-ceramide). PKC activity was determined by measuring the incorporation of [gamma- 32 P]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) 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.