6th ESACP Congress, Heidelberg, April 7-11, 1999

A050
IMAGING OF IN SITU DNA SENSITIVITY TO DENATURATION DURING THE CELL CYCLE AND APOPTOSIS BY CONFOCAL MICROSCOPY
Dobrucki J 1, Slupczynska A 1, Krzeszowiec W 1, Darzynkiewicz Z 2

1) Biophys. Dept., Jagiellonian Univ., Krakow, Poland, 2) New York Medical College, The Cancer Center Inst., N.Y., USA

Sensitivity of DNA in situ to denaturation correlates with changes in chromatin condensation and varies during the cell cycle. The metachromatic dye acridine orange (AO), which differentially stains double- versus single-stranded DNA was used as a marker of DNA denaturation. DNA in condensed chromatin of mitotic or apoptotic cells was shown to have higher sensitivity to denaturation in comparison with DNA in interphase cells. Flow cytometry or standard fluorescence microscopy, however, could not reveal DNA denaturability in interphase nuclei or along individual chromosomes. We have investigated green and red fluorescence of the AO-stained normal human fibroblasts, apoptotic HL-60 lymphoma cells as well as polytene chromosomes from insect larvae using confocal microscopy and deconvolution. Nuclei of interphase cells exhibited predominantly green fluorescence representing double-stranded DNA and small, distinct areas of red staining representing denatured DNA. The proportion of highly condensed DNA increased in cells approaching mitosis. Areas of highly condensed DNA were found in polytene chromosomes; regions of high transcriptional activity exhibited green fluorescence. Areas of green and red-staining DNA were found in nuclei of cells entering apoptosis and in apoptotic bodies. These studies provide new information about the structure of chromatin in interphase and mitotic cells and the stability of DNA helix in situ.