Immunocytochemical localization of transient DNA strand breaks in differentiating myotubes using in situ nick-translation☆

摘要

WehavelocalizedDNAstrandbreaksduringinvitrochickenmyogenesisbyrepairingnicksinnucleioffixedcellmonolayersinsituwithbiotin-11-dUTP,followedbyimmunocytochemicaldetectionofincorporatedbiotinwithrabbitanti-biotinandFITC-labeledgoatanti-rabbitantibodies.Noaccumulationsofbiotinsufficientforimmunocytochemicaldetectionwereobservedin23-hrculturesofdividingcells.In33-and43-hrcultures,biotinwasfirstdetectedinonly3%ofthenuclei,allofwhichappearedtobeinfusingmyoblastsorsmallmyotubes.Incontrast,culturesofyoung,highlyfusedmyotubes(56hr)exhibited18%biotinylatednuclei;virtuallyallofthesenuclei,mostofwhichweregroupedasaggregates,werewithinmyotubes.Inoldercultures(73and94hr)incorporationofbiotinintomyotubenucleimarkedlydecreased,whileincreaseswerenotedinnucleiofmononuclearcells.Theseresultsindicatethatextensivesingle-strandedDNAnickingoccursinnucleiofyoungmyotubes,followedbyrepairasterminaldifferentiationensues.