Indications for an active process underlying spontaneous and radiation-induced micronucleation in L929 cells.
OBJECTIVE
To investigate the mechanism of micronucleus formation in irradiated L929 cells.


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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Institute of Radiobiology, Federal Armed Forces Medical Academy, Munich, Germany. 101653.1373@compuserve.com
Frombeck
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Van Beuningen
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D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000818:Animals; D001312:Aurintricarboxylic Acid / Q000494:pharmacology / Q000031:analogs & derivatives; D001559:Benzhydryl Compounds / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000378:metabolism; D002455:Cell Division / Q000502:physiology / Q000528:radiation effects; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004273:DNA, Neoplasm / Q000378:metabolism / Q000528:radiation effects; D016521:Electrophoresis, Gel, Pulsed-Field; D004720:Endonucleases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005354:Fibrosarcoma; D051379:Mice; D048629:Micronuclei, Chromosome-Defective / Q000528:radiation effects*; D015162:Micronucleus Tests; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D013096:Spermine / Q000494:pharmacology; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013713:Teniposide / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors; D014158:Transcription, Genetic / Q000528:radiation effects; D014407:Tumor Cells, Cultured / Q000528:radiation effects*
D001559:Benzhydryl Compounds; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; C064769:N-acetylsphingosine; D018809:Proliferating Cell Nuclear Antigen; D059005:Topoisomerase II Inhibitors; D013096:Spermine; D001312:Aurintricarboxylic Acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C017037:aurin; D013713:Teniposide; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D004720:Endonucleases; D001973:Bromodeoxyuridine; D013110:Sphingosine