APOPTOTIC CELL-NUCLEI FAVOR AGGREGATION AND FLUORESCENCE QUENCHING OFDNA DYES

Citation
J. Erenpreisa et al., APOPTOTIC CELL-NUCLEI FAVOR AGGREGATION AND FLUORESCENCE QUENCHING OFDNA DYES, HISTOCHEM C, 108(1), 1997, pp. 67-75
Citations number
24
Categorie Soggetti
Cell Biology",Microscopy
Journal title
HISTOCHEMISTRY AND CELL BIOLOGY
ISSN journal
09486143 → ACNP
Volume
108
Issue
1
Year of publication
1997
Pages
67 - 75
Database
ISI
SICI code
0948-6143(1997)108:1<67:ACFAAF>2.0.ZU;2-9
Abstract
Apoptotic cell nuclei are known to stain hyperchromatically with absor ption dyes and dimly with many DNA fluorochromes. We hypothesised that both optical phenomena have the same cause - the ability of apoptotic chromatin to aggregate cationic dyes. This hypothesis was tested usin g prednisolone-primed rat thymus, which is known to contain apoptotic cells. The apoptotic cells were classified as early and late, based on their morphology, in thin and semithin sections and in thymus imprint s on slides. Direct reaction for DNA strand breaks (TUNEL) indicated t he presence of breaks in both categories of cells, with more intense l abelling in late apoptosis. The chromatin ultrastructure of early apop totic cells initially retained the supranucleosomal order of packaging which characterises control cells, whereas the dense chromatin of lat e apoptotic cells possessed the degraded structure. Absorption spectra of the toluidine blue-stained early apoptotic cell chromatin revealed a metachromatic shift, indicating a change of DNA conformation and po lymerisation of the dye. When the staining was performed by acridine o range (preceded by a short acid treatment), a paradoxical several-fold increase of fluorescence intensity at a several-fold dilution of the dye was found. The simultaneous reduction of the ratio of red to green components of fluorescence confirmed that the concentration-dependent fluorescence quenching was due to aggregation of the dye. The results suggest that the enhanced affinity of the chromatin of early apoptoti c cells for cationic dyes is associated with conformational relaxation rather than degradation of DNA. In late apoptotic cells, the very den se packaging of degraded DNA promotes further aggregation of dyes. The results suggest alternative methods for detection and discrimination of early and late apoptotic cells.