Nuclear apoptosis detection by flow cytometry: influence of endogenous endonucleases.
Nuclear apoptosis is characterized by chromatin condensation and progressive DNA cleavage into high-molecular-weight fragments and oligonucleosomes. These complex phenomena can be mediated by the activation of a multiplicity of enzymes, characterized by specific patterns of cation dependance, pH requirement, and mode of activation. The significance of this multiplicity of enzymes that cleave genomic DNA has been attributed to the need of death effector pathways specific for cell types/tissues, the level of cell differenciation, and the nature of the apoptotic stimuli. The activation of these factors contributes to the development of alterations that can be detected specifically by flow cytometric assays, namely, propidium iodide assays, acridine orange/ethidium bromide double staining, the TUNEL and ISNT techniques, and the assays of DNA sensitivity to denaturation. Although applicable to a wide spectrum of cell types, an increasing body of literature indicates that these techniques cannot be universally applied to all cell lines and apoptotic conditions: The requirement of a particular mediator(s) of nuclear apoptosis or the absence of endonuclease activity can limit the relevance of certain techniques. Finally, endonucleases recruited during primary necrosis can introduce nuclear alterations detected by some assays and raise the problem of their specificity. This review underlines the need for strategies to accurately detect and quantify nuclear apoptosis by flow cytometry when new cell systems and apoptotic conditions are considered.
Lecoeur
Hervé
H
Theraptosis Research Laboratory, Theraptosis S.A. Pasteur Biotop, Institut Pasteur, Paris, France.
D000818:Animals; D017209:Apoptosis; D002467:Cell Nucleus; D053938:DNA Fragmentation; D004720:Endonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D005434:Flow Cytometry / Q000379:methods; D006801:Humans
D004720:Endonucleases