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The behaviour of nuclear domains in the course of apoptosis
1Istituto di Morfologia Umana Normale, Università di Chieti, Italy.
Abstract:
Programmed cell death is activated, by different stimuli and in many cell types, to regulate cell population balance during tissue proliferation and embryogenesis. Its initial event seems to be, in most cases, the activation of a Ca(2+)-dependent endonuclease, causing DNA cleavage into nucleosomic fragments. Its morphological expression is characterized by deep nuclear changes, consisting of typical cap-shaped chromatin marginations, followed by nuclear fragmentation and final formation of numerous micronuclei. Cytoplasmic damage appears in a very late stage of the process and the greatest part of the phenomenon appears to take place despite good preservation of the plasma membrane and organellar component. In the present study we analyzed apoptosis in camptothecin-treated HL60 leukaemia cells, and in freshly isolated mouse thymocytes treated with dexamethasone. The process was first quantified and time monitored by flow cytometry. Subsequently the specimens were processed for morphological examination in order to investigate the behaviour of the different nuclear domains. To follow DNA and RNA localization, we utilized osmium ammine and DNase-colloidal gold cytochemical reactions. The concentration of most DNA in the cap-shaped structures was demonstrated by these reactions. Confocal microscopy of cells processed by in situ nick-translation suggested that DNA was firstly cleaved and subsequently condensed in cup-shaped structures. Despite the strong nuclear modifications, nucleoli could be clearly recognized until the late apoptotic stages.
Insights
Programmed cell death, or apoptosis, involves DNA fragmentation and nuclear changes. This study shows DNA cleavage and condensation into specific nuclear structures during apoptosis in leukemia and thymocyte cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Programmed cell death (apoptosis) is crucial for tissue homeostasis and development.
- Early events in apoptosis often involve calcium-dependent endonuclease activation and DNA cleavage.
- Morphological hallmarks include nuclear changes like chromatin margination and fragmentation.
Purpose of the Study:
- To investigate the morphological and molecular events of apoptosis in specific cell models.
- To analyze the behavior of nuclear domains during programmed cell death.
- To elucidate the localization and dynamics of DNA during apoptosis.
Main Methods:
- Flow cytometry for apoptosis quantification and time monitoring.
- Morphological examination of cellular and nuclear structures.
- Cytochemical reactions (osmium ammine, DNase-colloidal gold) for DNA/RNA localization.
- In situ nick-translation and confocal microscopy for DNA cleavage and condensation analysis.
Main Results:
- Apoptosis was induced in HL60 leukaemia cells (camptothecin) and mouse thymocytes (dexamethasone).
- DNA was found to concentrate in cap-shaped structures, indicating cleavage and subsequent condensation.
- Nuclear modifications occurred while preserving plasma membrane and organelles until late stages.
- Nucleoli remained recognizable throughout the apoptotic process.
Conclusions:
- Apoptosis involves specific DNA cleavage and condensation into distinct nuclear structures.
- The process occurs with significant nuclear restructuring but relative preservation of cytoplasmic components.
- Understanding these nuclear dynamics provides insights into programmed cell death mechanisms.