The Schrödinger's cat quandary in cell biology: integration of live cell functional assays with measurements of fixed cells in analysis of apoptosis.
The existing cytometric methodologies do not allow one to directly correlate, within the same cells, functional cell attributes that are revealed supravitally with features that require cell fixation to be detected or measured. Taking advantage of the "file merge" feature of the laser-scanning cytometer, we have been able to correlate the supravital changes that occur during apoptosis, namely the drop in mitochondrial transmembrane potential (Delta Psim) and generation of the reactive oxygen intermediates (ROIs), with features revealed by analysis of fixed cells: the cell cycle position and DNA fragmentation. The cell cycle position was established based on the cell's stainability with propidium iodide while DNA fragmentation was assessed by in situ DNA strand break labeling using exogenous terminal deoxynucleotidyltransferase. During apoptosis of HL-60 cells induced by the DNA topoisomerase I inhibitor camptothecin (CPT), the dissipation of Delta Psim occurred preferentially in S-phase cells and preceded the appearance of DNA strand breaks. Essentially all cells with DNA strand breaks had dissipated Delta Psim. Compared to the decrease of Delta Psim, the CPT-induced rise in ROIs during apoptosis was less restricted to S-phase cells. Furthermore, no elevation of ROIs was detected in a significant proportion of cells with DNA strand breaks. The data suggest that DNA fragmentation may occur in some cells prior to the increase in ROIs and thus, unlike the dissipation of Delta Psim, the oxidative stress may not be a prerequisite for activation of an endonuclease. Alternatively, the oxidative stress may be a transient event, occupying a narrow "time window" during the apoptotic process. The approach opens a possibility to study direct relationships, within the same cells, between cellular changes (e.g., occurring during apoptosis, mitogenesis, differentiation, etc.) detected by functional assays of live cells and changes that cannot be analyzed supravitally.
Li
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The Brander Cancer Research Institute, New York Medical College, Valhalla, New York, 10595, USA.
Darzynkiewicz
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D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001681:Biological Assay / Q000379:methods*; D002166:Camptothecin / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D019044:Image Cytometry / Q000379:methods; D016707:Tissue Fixation
D002166:Camptothecin