![]() |
6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A050
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.
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