THE BEHAVIOR OF NUCLEAR DOMAINS IN THE COURSE OF APOPTOSIS

Programmed cell death is activated, by different stimuli and in many c ell types, to regulate cell population balance during tissue prolifera tion and embryogenesis. Its initial event seems to be, in most cases, the activation of a Ca2+-dependent endonuclease, causing DNA cleavage into nucleosomic fragments. Its morphological expression is characteri zed by deep nuclear changes, consisting of typical cap-shaped chromati n marginations, followed by nuclear fragmentation and final formation of numerous micronuclei. Cytoplasmic damage appears in a very late sta ge of the process and the greatest part of the phenomenon appears to t ake place despite good preservation of the plasma membrane and organel lar component. In the present study we analyzed apoptosis in camptothe cin-treated HL60 leukaemia cells, and in freshly isolated mouse thymoc ytes treated with dexamethasone. The process was first quantified and time monitored by flow cytometry. Subsequently the specimens were proc essed for morphological examination in order to investigate the behavi our of the different nuclear domains. To follow DNA and RNA localizati on, we utilized osmium ammine and DNase-colloidal gold cytochemical re actions. The concentration of most DNA in the cap-shaped structures wa s demonstrated by these reactions. Confocal microscopy of cells proces sed by in situ nick-translation suggested that DNA was firstly cleaved and subsequently condensed in cup-shaped structures. Despite the stro ng nuclear modifications, nucleoli could be clearly recognized until t he late apoptotic stages.